EP2007423A2 - Ctla4 antibody combination therapy - Google Patents

Ctla4 antibody combination therapy

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Publication number
EP2007423A2
EP2007423A2 EP20070734181 EP07734181A EP2007423A2 EP 2007423 A2 EP2007423 A2 EP 2007423A2 EP 20070734181 EP20070734181 EP 20070734181 EP 07734181 A EP07734181 A EP 07734181A EP 2007423 A2 EP2007423 A2 EP 2007423A2
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Prior art keywords
cancer
antibody
agent
anti
administered
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EP20070734181
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German (de)
French (fr)
Inventor
Jesus Gomez-Navarro
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Pfizer Products Inc
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Pfizer Products Inc
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507Comprising a combination of two or more separate antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Abstract

The invention relates to administration of an anti-CTLA4 antibody, particularly human antibodies to human CTLA4, such as those having amino acid sequences of antibodies 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, CP-675,206 (11.2.1), and ipilimumab, in combination with a therapeutic agent for treatment of cancer. An exemplary method of the invention comprises administering an anti-CTLA4 antibody, preferably, CP-675,206, and a chemotherapeutic agent, preferably, gemcitabine, for treatment of pancreatic cancer, among other treatment modalities.

Description

CTLA4 ANTIBODY COMBINATION THERAPY

Background of the Invention

The present invention relates to uses and compositions comprising an anti-CTLA4 antibody and at least one therapeutic agent for treatment of cancer. The invention further relates to administering, in addition to the antibody-therapeutic agent inhibitor combination, at least one additional therapy such as, among others, additional therapeutic agents, radiation and stem cell transplant.

Current antitumor agents act by a variety of mechanisms that inhibit cancer cell growth and division, ultimately destroying the malignant cell. However, because these cytotoxic agents are generally not selective for neoplastic cells, they destroy normal cells, disrupt physiologic functions, and are often associated with adverse effects. An alternative approach to cancer therapy is to target the immune system ("immunotherapy") rather than the tumor itself so that the patient's own immune system attacks tumors while sparing non-tumor cells. One cancer immunotherapy approach targets cytotoxic T lymphocyte-associated antigen 4 (CTLA4; CD152), which is a cell surface receptor expressed on activated T cells. Binding of CTLA4 to its natural ligands, B7.1 (CD80) and B7.2 (CD86), delivers a negative regulatory signal to T cells, and blocking this negative signal results in enhanced T cell immune function and antitumor activity in animal models (Thompson and Allison Immunity 7:445-450 (1997); McCoy and LeGros lmmunol.& Cell Biol. 77:1-10 (1999)). Several studies have demonstrated that CTLA4 blockade using antibodies markedly enhances T cell- mediated killing of tumors and can induce antitumor immunity (see, e.g., Leach et al., Science 271:1734-1736 (1996); Kwon et al. Proc. Natl. Acad. ScL USA 94:8099-8103 (1997); Kwon et al., Natl. Acad, Sci. USA 96:15074-15079 (1999); Yang et al. Cancer Res 57:4036-41 (1997); Hurwitz et al. Proc. Natl. Acad. Sci. USA 95:10067-71 (1998)). CTLA4 antibodies and their uses are described in, e.g., the following applications and patents: U.S. Patent Application No. 09/472,087, now issued as U.S. Patent No. 6,682,736; Int. Appl. No. PCT/US99/30895 (published June 29, 2000, as WO 00/37504); U.S. Pat. Appl. No. 10/612,497 (published November 18, 2004, as US 2004/0228858); U.S. Pat. Appl. No. 10/776,649 (published November 18, 2004, as US 2004/0228861); Int. Appl. No. Int. Appl. No. PCT/USOO/23356 (published March 1 , 2001, as WO 01/14424) (e.g., antibody 10D1 , also known as MDX-010, and ipilimumab, Medarex, Princeton, NJ); Int. Appl. No. PCT/US99/28739 (published June 8, 2000, as WO 00/32231); U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,207,156; U.S. Pat. No. 5,844,095, to Linsley et al.; Int. Appl. No. PCT/US92/05202 (published Jan. 7, 1993, as WO 93/00431); U.S. Patent Appl. No. 10/153,382 (published May 8, 2003, as US 2003/0086930); U.S. Pat. Appl. No. 10/673,738 (published February 24, 2005 as US 2005/0042223); U.S. Pat. Appl. No. 11/085,368 (published October 13, 2005, as US 2005/0226875); U.S. Pat. Appl. No. 60/624,856 (filed Nov. 4, 2004), now published as WO2006/048749, May 11 , 2006; U.S. Pat. Appl. No. 60/664,364 (filed March 23, 2005) and U.S. Pat. Appl. No. 60/711 ,707 (filed Aug. 26, 2005) now published as WO 2006/101691 Sept. 28, 2006; U.S. Pat. Appl. No. 60/664,653 (filed March 23, 2005) now published as WO 2006/101692, Sept. 28, 2006; U.S. Pat. Appl. No. 60/697,082 (filed July 7, 2005) now WO 2007/008463 published Jan. 18, 2007.

Despite the successes of currently available anti-cancer treatments, complete responses to these treatments or prolonged survival are infrequently observed, and the patient population refractory to these treatments is still large. Thus, there is an unmet need for the development of new therapeutic regimens, particularly those capable of augmenting or potentiating the anti-tumor activity of other anti-neoplastic agents while reducing the cytotoxic side effects of current chemotherapeutics, and the present invention meets this need.

Summary of the Invention The present invention includes a method for the treatment of cancer in a patient in need of such treatment. The method comprises administering to the patient a therapeutically effective amount of an anti-CTLA4 antibody, e.g., ipilimumab (also referred to as MDX-010) and CP-675,206 (also referred to as 11.2.1. and ticilimumab), in combination with a therapeutically effective amount of at least one therapeutic agent, wherein the cancer and the agent are selected from the group consisting of:

(a) the cancer is non-Hodgkin's lymphoma (NHL) and wherein the agent is rituximab;

(b) the cancer is NHL and the agent is cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP);

(c) the cancer is NHL and the agent is cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (CHOP-R);

(d) the cancer is lung cancer and the agent is bevacizumab;

(e) the cancer is non-small cell lung cancer (NSCLC) and the agent is gefitinib;

(f) the cancer is NSCLC and the agent is bevacizumab;

(g) the cancer is NSCLC and the agent is a taxane and gemcitabine, and further wherein the taxane is selected from the group consisting of docetaxel and paclitaxel;

(h) the cancer is NSCLC and the agent is a taxane and a platinum compound; (i) the cancer is NSCLC and the agent is docetaxel; 0) the cancer is NSCLC and the agent is erlotinib; (k) the cancer is NSCLC and the agent is pemetrexed; (I) the cancer is NSCLC and the agent is a platinum compound;

(m) the cancer is gastric cancer and the agent is irinotecan; (n) the cancer is gastric cancer and the agent is fluorouracil and leucovorin;

(o) the cancer is liver cancer and the agent is doxorubicin, ifosfamide and vincristine;

(p) the cancer is liver cancer and the agent is doxorubicin and vincristine;

(q) the cancer is colorectal carcinoma (CRC) and the agent is fluorouracil; (r) the cancer is CRC and the agent is capecitabine;

(s) the cancer is CRC and the agent is fluorouracil, leucovorin, and oxaliplatin (FOLFOX);

(t) the cancer is CRC and the agent is fluorouracil, leucovorin, and irinotecan (FOLFIRI); (u) the cancer is CRC and the agent is cetuximab;

(v) the cancer is chronic myeloid leukemia (CML) and the agent is imatinib mesylate;

(w) the cancer is chronic lymphocytic leukemia (CLL) and the agent is imatinib mesylate;

(x) the cancer is pancreatic cancer and the agent is gemcitabine; (y) the cancer is breast cancer and the agent is a taxane;

(z) the cancer is breast cancer and the agent is cyclophosphamide, doxorubicin and a taxane;

(aa) the cancer is breast cancer and the agent is selected from the group consisting of tamoxifen, anastrazole, letrozole, and fulvestrant; (bb) the cancer is breast cancer and the agent is trastuzumab;

(cc) the cancer is breast cancer and the agent is bevacizumab;

(dd) the cancer is breast cancer and the agent is cetuximab;

(ee) the cancer is breast cancer and the agent is axitinib;

(ff) the cancer is bladder cancer and the agent is Bacillus Calmette-Guerin (BCG); (gg) the cancer is bladder cancer and the agent is gemcitabine and cisplatin;

(hh) the cancer is melanoma and the agent is interferon alpha;

(ii) the cancer is multiple myeloma and the agent is bortezomib;

(jj) the cancer is multiple myeloma and the agent is dexamethasone and thalidomide; and (kk) the cancer is ovarian cancer and the agent is carboplatin and paclitaxel.

In one aspect, the treatment is selected from the group consisting of neoadjuvant therapy, adjuvant therapy, first-line therapy, second-line therapy, and third-line therapy.

In another aspect, the agent is administered sequentially or contemporaneously with the antibody. In yet another aspect, the taxane is paclitaxel and the platinum compound is carboplatin.

In one aspect, the method further comprises administering at least one agent selected from the group consisting of bevacizumab, PF03512676, and sunitinib. In another aspect, the method further comprises administering at least one agent selected from the group consisting of erlotinib and pemetrexed, wherein the treatment comprises second line therapy.

In one aspect, the therapeutically effective amount of the antibody ranges from about 1 mg/kg to 40 mg/kg. In another aspect, the therapeutically effective amount of the antibody ranges from about 3 mg/kg to 15 mg/kg.

In one aspect, the NHL is indolent NHL and the treatment comprises first line therapy.

In another aspect, the NHL is aggressive NHL and the treatment comprises second line therapy. In a further aspect, the treatment comprises first line therapy.

In one aspect, the cancer is CRC and the agent is capecitabine and the treatment comprises first line therapy.

In another aspect, the cancer is CRC and the agent is FOLFOX and the treatment is selected from the group consisting of first line therapy and adjuvant therapy following surgical resection of a primary colon tumor.

In one aspect, the cancer is CML and the agent is imatinib mesylate and the treatment comprises first line therapy.

In another aspect, the cancer is CLL and the agent is imatinib mesylate and the treatment comprises first line therapy. In a further aspect, the cancer is pancreatic cancer and the agent is gemcitabine and the pancreatic cancer is selected from the group consisting of non-resectable Stage II, locally advanced Stage III, and metastatic Stage IV and wherein the treatment comprises first line therapy.

In another aspect, the cancer is breast cancer and the agent is a taxane and the treatment comprises first line therapy.

In one aspect, the cancer is ovarian cancer and the agent is carboplatin and paclitaxel and the treatment comprises first line therapy.

The invention includes a kit for the treatment of NSCLC comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of a carboplatin; a therapeutically effective amount of paclitaxel; an applicator; and an instructional material for the use of the kit. In one aspect, the kit further comprises a therapeutically effective amount of at least one agent selected from the group consisting of bevacizumab, sunitinib, and PF03512676.

The invention includes a kit for the treatment of NSCLC comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of an agent selected from the group consisting of docetaxel, erlotinib and pemetrexed; an applicator; and an instructional material for the use of the kit.

The invention includes a kit for the treatment of CRC comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of carboplatin; a therapeutically effective amount of paclitaxel; an applicator; and an instructional material for the use of the kit.

The invention includes a kit for the treatment of CRC comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of a fluorouracil; a therapeutically effective amount of leucovorin; a therapeutically effective amount of oxaliplatin; an applicator; and an instructional material for the use of the kit. The invention includes a kit for the treatment of pancreatic cancer comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of gemcitabine; an applicator; and an instructional material for the use of the kit.

The invention includes a kit for the treatment of ovarian cancer comprising a therapeutically effective amount of CP-675,206; a therapeutically effective amount of carboplatin; a therapeutically effective amount of paclitaxel; an applicator; and an instructional material for the use of the kit.

The invention includes a method for preventing or treating infection by HIV or for preventing, treating or delaying the onset of AIDS in a patient in need thereof. The method comprises administering to the patient a therapeutically effective amount of anti-CTLA4 antibody CP-675,206 and further comprises administering a therapeutically effective amount of at least one antiviral agent selected from the group consisting of an HIV protease inhibitor, a non-nucleoside reverse transcriptase inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, a CCR5 antagonist, an inhibitor of gp120 interaction with CD4, an HIV fusion inhibitor, a HIV integrase inhibitor, an RNaseH inhibitor, a prenylation inhibitor, and a maturation inhibitor.

In one aspect, the CCR5 antagonist is maraviroc.

In another aspect, the method further comprises assessing the co-receptor tropism of the HIV.

The invention includes a method for preventing or treating infection by HIV or for preventing, treating or delaying the onset of AIDS in a patient in need thereof, where the method comprises administering to the patient a therapeutically effective amount of an anti- CTLA4 antibody and maraviroc.

In one aspect, the anti-CTLA4 antibody is selected from the group consisting of CP- 675,206 and ipilimumab.

Alternate embodiments of the invention are described below, such as those employing alternate anti-CTLA4 antibodies and involving different methods.

Brief Description of the Drawings The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention there are shown in the drawings embodiment(s) which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

Figure 1 , comprising Figures 1A-1D, shows the nucleotide and amino acid sequences of anti-CTLA4 antibody 4.1.1. Figure 1A shows the full length nucleotide sequence for the 4.1.1 heavy chain (SEQ ID NO:1). Figure 1 B shows the full length amino acid sequence for the 4.1.1 heavy chain (SEQ ID NO:2), and the amino acid sequence for the 4.1.1 heavy chain variable region (SEQ ID NO:3) designated between brackets "[ ]". The amino acid sequence of each 4.1.1 heavy chain CDR is underlined. The CDR sequences are as follows: CDR1 : GFTFSSHGMH (SEQ ID NO:4); CDR2: VIWYDGRNKYYADSV (SEQ ID NO:5); and CDR3: GGHFGPFDY (SEQ ID NO:6). Figure 1C shows the nucleotide sequence for the 4.1.1 light chain (SEQ ID NO:7). Figure 1D shows the amino acid sequence of the full length 4.1.1 light chain (SEQ ID NO:8), and the variable region as indicated between brackets "[ ]" (SEQ ID NO:9). The amino acid sequence of each CDR is indicated as follows: CDR1 : RASQSISSSFLA (SEQ ID NO:10); CDR2: GASSRAT (SEQ ID NO:11); and CDR3: QQYGTSPWT (SEQ ID NO:12).

Figure 2, comprising Figures 2A-2D, shows the nucleotide and amino acid sequences of anti-CTLA4 antibody 4.13.1. Figure 2A shows the full length nucleotide sequence for the 4.13.1 heavy chain (SEQ ID NO: 13). Figure 2B shows the full length amino acid sequence for the 4.13.1 heavy chain (SEQ ID NO: 14), and the amino acid sequence for the 4.13.1 heavy chain variable region (SEQ ID NO:15) designated between brackets "[ ]". The amino acid sequence of each 4.13.1 heavy chain CDR is underlined. The CDR sequences are as follows: CDR1: GFTFSSHGIH (SEQ ID NO:16); CDR2: VIWYDGRNKDYADSV (SEQ ID NO: 12); and CDR3: VAPLGPLDY (SEQ ID NO: 18). Figure 2C shows the nucleotide sequence for the 4.13.1 light chain (SEQ ID NO:19). Figure 2D shows the amino acid sequence of the full length 4.13.1 light chain (SEQ ID NO:20), and the variable region as indicated between brackets "[ ]" (SEQ ID NO:21 ). The amino acid sequence of each CDR is indicated as follows: CDR1 : RASQSVSSYLA (SEQ ID NO:22); CDR2: GASSRAT (SEQ ID NO:23); and CDR3: QQYGRSPFT (SEQ ID NO:24).

Figure 3, comprising Figures 3A-3D, shows the nucleotide and amino acid sequences of anti-CTLA4 antibody CP-675,206. Figure 3A shows the full length nucleotide sequence for the CP-675,206 heavy chain (SEQ ID NO:25). Figure 3B shows the full length amino acid sequence for the CP-675,206 heavy chain (SEQ ID NO:26), and the amino acid sequence for the CP-675,206 heavy chain variable region (SEQ ID NO:27) designated between brackets "[ ]". The amino acid sequence of each CP-675,206 heavy chain CDR is underlined. The CDR sequences are as follows: CDR1 : GFTFSSYGMH (SEQ ID NO:28); CDR2: VIWYDGSNKYYADSV (SEQ ID NO:29); and CDR3: DPRGATLYYYYYGMDV (SEQ ID NO:30). Figure 3C shows the nucleotide sequence for the CP-675,206 light chain (SEQ ID NO:31). Figure 3D shows the amino acid sequence of the full length CP-675,206 light chain (SEQ ID NO:32), and the variable region as indicated between brackets "[ ]" (SEQ ID NO:33). The amino acid sequence of each CDR is indicated as follows: CDR1 : RASQSINSYLD (SEQ ID NO:34); CDR2: AASSLQS (SEQ ID NO:35); and CDR3: QQYYSTPFT (SEQ ID NO:36).

Figure 4 is a graph depicting the plasma serum levels in Rhesus monkeys following exposure to an anti-CTLA4 antibody. All Rhesus monkeys were administered influenza vaccine FLUZONE intramuscularly (IM) on week 0 and again on week 4. In addition to FLUZONE, animals in Group 1 (animal number 1 (closed circle), 2 (closed square), and 3 (closed triangle)) received a single dose of anti-CTL4 antibody 4.1.1 (also referred to as CP- 642,570) at 5 mg/kg intravenously (IV) on week 0. Animals in Group 3 (animal number 4 (open circle), 5 (open square), 6 (open triangle), and 7 (marked by "x")) received a single dose of an irrelevant human antibody (anti-KLH antibody) at 5 mg/kg IV on week 0.

Figure 5 is a graph depicting the serum level of a neopterin, a marker which is suggestive of immune activity, in Rhesus monkeys immunized with FLUZONE IM on week 0 and week 4 and administered anti-CTLA4 antibody 4.1.1 at 5 mg/kg IV on week 0. Animal number AG40 (closed diamond), number AK80 (closed square) and number AM90 (closed triangle) were in group 1 and received anti-CTLA4 antibody and FLUZONE on day 0 followed by FLUZONE on week 0. Animal number AK76 (x), number AM84 (*), and number BC13 (open circle) were in control group 2 and were administered FLUZONE on week 0 and week 4 and also administered an irrelevant human antibody (anti-KLH antibody) at 5 mg/ml IV on week 0 of the protocol. Figure 6 is a graph depicting the level of a 2-5 adenylate synthetase in white blood cell pellets isolated from the blood of Rhesus monkeys immunized with FLUZONE IM on weeks 0 and 4 and further administered anti-CTLA4 antibody 4.1.1 (5mg/kg IV) on week 0. Peripheral blood cells were obtained from each animal in each group 1 (FLUZONE IM weeks 0 and 4 and anti-CTLA4 antibody week 0) and group 2 (FLUZONE IM on weeks 0 and 4 and an irrelevant antibody (anti-KLH 5 mg/kg IV) administered on week 0) at week 2 (light gray shading), week 4 (darker gray shading) and week 6 (unshaded bars). Where the values exceeded 4000 pmol/dl, the values are shown in each bar. The cells were pelleted and the level of 2-5 adenylate synthetase in the pellets obtained from each animal was assessed. Figure 7 is a graph depicting the IgG anti-FLUZONE titer in Rhesus monkeys administered anti-CTLA4 antibody. Serum anti-FLUZONE IgG titers were assessed for animals in Group 1 (open triangle; administered FLUZONE on weeks 0 and 4 and administered anti-CTLA4 antibody on week 0), Group 2 (open square; administered FLUZONE on weeks 0 and 4 and administered an irrelevant human anti-KLH antibody on week 0), Group 3 (closed circle; administered FLUZONE on weeks 0 and 4 and administered anti-CTLA4 antibody on week 4), Group 4 (open circle; administered FLUZONE on weeks 0 and 4 and administered an irrelevant human anti-KLH antibody on week 4) and Group 5 (closed triangle; administered FLUZONE on weeks 0 and 4 but no antibody was administered). FLUZONE was administered IM and antibodies were administered IV at 5 mg/kg. Anti-FLUZONE IgG titers were assessed one week before immunization with FLUZONE (week -1, also referred to as "prebleed") and then at week 0, week 2, week 4, week 6 and week 8. Animals in Group 3 (immunized with FLUZONE on weeks 0 and 4 and administered anti-CTLA4 antibody on week 4) demonstrated increased anti-FLUZONE IgG serum titers compared with animals in the other groups. Figure 8 is a graph depicting the anti-FLUZONE IgG serum titer in Rhesus monkeys immunized with FLUZONE and on weeks 0 and 4 and administered anti-CLTA4 antibody on week 0 (Group 1), immunized with FLUZONE on weeks 0 and 4 and administered an irrelevant antibody on week 0 (Group 2), immunized with FLUZONE on weeks 0 and 4 and administered anti-CTLA4 antibody on week 4 (Group 3), immunized with FLUZONE on weeks 0 and 4 and administered an irrelevant antibody on week 0 (Group 4), and immunized with FLUZONE on weeks 0 and 4 (Group 5). The data points are separated by animal group and the titers for each individual animal within each group for the following data points: prebleed (week -1 ; +), week 0 (open triangle), week 2 (closed triangle), week 4 (open squares), week 6 (closed circle) and week 8 (open circle). Figure 9 is a bar graph depicting the anti-FLUZONE IgG antibody titers at week 6 in

Rhesus monkeys immunized with FLUZONE on weeks 0 and 4 (Groups 1-5). Group 1 animals received anti-CTLA4 (5 mg/kg IV) on week 0. Group 2 animals received irrelevant antibody anti-KLH (5 mg/kg IV) on week 0. Group 3 animals received anti-CTLA4 (5 mg/kg IV) on week 4. Group 4 animals received irrelevant antibody anti-KLH (5 mg/kg IV) on week 4. Group 5 animals received only FLUZONE immunizations on weeks 0 and 4.

Detailed Description Of The Invention

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

The methods and techniques of the present invention are generally performed according to methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Such references include, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

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, Methods MoI. Biol. 243:307-31 (1994).

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: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: 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., Science 256:1443-45 (1992), herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Preferred amino acid substitutions are those which: (1 ) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs comprising substitutions, deletions, and/or insertions can include various muteins of a sequence other than the specified peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the specified sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts, e.g., outside of the CDRs). A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991), which are each incorporated herein by reference.

Sequence similarity for polypeptides 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, Genetics Computer Group (GCG available from Genetics Computer Group, Inc.), also referred to as the Wisconsin Package, is an integrated software package of over 130 programs for accessing, analyzing and manipulating nucleotide and protein sequences. GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence similarity, homology and/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, version 7.0, version 9.1 , and version 10.0.

Polypeptide sequences also can be compared using FASTA, a program in GCG, using default or recommended parameters. 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, Methods Enzymol. 183:63-98 (1990); Pearson, Methods MoI. Biol. 132:185-219 (2000)). 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., J. MoI. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Each heavy chain is comprised of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of lmmunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia & Lesk, J. MoI. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CTLA4). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (Hi) a Fd fragment consisting of the Vμ and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. ScL USA 85:5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl. Acad. ScL USA 90:6444-6448 (1993); Poljak et al. Structure 2:1121-1123 (1994)).

Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. Human Antibodies and Hybridomas 6:93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. MoI. Immunol. 31:1047-1058 (1994)). Other examples include where one or more CDRs from an antibody are incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that specifically binds to an antigen of interest, such as CTLA4. In such embodiments, the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

Where an "antibody" is referred to herein with respect to the present invention, it should be understood that an antigen-binding portion thereof may also be used. An antigen- binding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. In embodiments having one or more binding sites, the binding sites may be identical to one another or may be different.

The terms "human antibody" or "human sequence antibody", as used interchangeably herein, include antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The human sequence 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). However, the term "human antibody", as used herein, is not intended to include "chimeric" antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized" or PRI MATIZED ™ antibodies).

The term "chimeric antibody" as used herein means an antibody that comprises regions from two or more different antibodies. In one embodiment, one or more of the CDRs are derived from a human anti-CTLA4 antibody. In another embodiment, all of the CDRs are derived from a human anti-CTLA4 antibody. In another embodiment, the CDRs from more than one human anti-CTLA4 antibodies are combined in a chimeric human antibody. For instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human anti- CD40 antibody, a CDR2 from the light chain of a second human anti-CTLA4 antibody and a CDR3 and CDR3 from the light chain of a third human anti-CTLA4 antibody, and the CDRs from the heavy chain may be derived from one or more other anti-CD40 antibodies. Further, the framework regions may be derived from one of the same anti-CTLA4 antibodies or from one or more different human(s).

Moreover, as discussed previously herein, chimeric antibody includes an antibody comprising a portion derived from the germline sequences of more than one species. "Glycoform" refers to a complex oligosaccharide structure comprising linkages of various carbohydrate units. Such structures are described in, e.g., Essentials of Glycobiology Varki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also provides a review of standard glycobiology nomenclature. Such glycoforms include, but are not limited to, G2, G1 , GO, G-1 , and G-2 (see, e.g., International Patent Publication No. WO 99/22764).

"Glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein (e.g., the glycoform) as well as to the site(s) to which the glycoform(s) are covalently attached to the peptide backbone of a protein, more specifically to an immunoglobulin protein. It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycoforms and/or glycosylation patterns compared with each other. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein are part of the instant invention, regardless of the glycosylation of the antibodies. By the term "effective amount" , or "therapeutically effective amount," as used herein, is meant an amount that when administered to a mammal, preferably a human, mediates a detectable therapeutic response compared to the response detected in the absence of the compound. A therapeutic response, such as, but not limited to, inhibition of and/or decreased tumor growth, tumor size, metastasis, and the like, can be readily assessed by a plethora of art-recognized methods, including, e.g., such methods as disclosed herein.

The skilled artisan would understand that the effective amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the stage of the disease, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

By the term "compete", as used herein with regard to an antibody, is meant that a first antibody, or an antigen-binding portion thereof, competes for binding with a second antibody, or an antigen-binding portion thereof, where binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s). For instance, cross-competing antibodies can bind to the epitope, or potion of the epitope, to which the antibodies used in the invention bind. Use of both competing and cross-competing antibodies is encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof, and the like), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. "Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compound, combination, and/or composition of the invention in the kit for affecting, alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell, a tissue, or a mammal, including as disclosed elsewhere herein.

The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.

Except when noted, the terms "patient" or "subject" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as veterinary subjects such as rabbits, rats, and mice, and other animals. Preferably, patient refers to a human. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

By the phrase "specifically binds," as used herein, is meant a compound, e.g., a protein, a nucleic acid, an antibody, and the like, which recognizes and binds a specific molecule, but does not substantially recognize or bind other molecules in a sample. For instance, an antibody or a peptide inhibitor which recognizes and binds a cognate ligand (e.g., an anti-CTLA4 antibody that binds with its cognate antigen, CTLA4) in a sample, but does not substantially recognize or bind other molecules in the sample. Thus, under designated assay conditions, the specified binding moiety (e.g., an antibody or an antigen-binding portion thereof) binds preferentially to a particular target molecule and does not bind in a significant amount to other components present in a test sample. A variety of assay formats may be used to select an antibody that specifically binds a molecule of interest. For example, solid- phase ELISA immunoassay, immunoprecipitation, BIAcore, FACS, and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with CTLA4. Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background, even more specifically, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (KD) is ≤ 1 μM, preferably ≤ 100 nM and most preferably ≤ 10 nM. The term "K0" refers to the equilibrium dissociation constant of a particular antibody- antigen interaction.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species (e.g., an anti-CTLA4 antibody) comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

As used herein, to "treat" means reducing the frequency with which symptoms of a disease (i.e., tumor growth and/or metastasis, or other effect mediated by the numbers and/or activity of immune cells, and the like) are experienced by a patient. The term includes the administration of the compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. "Combination therapy" embraces the administration of an immunostimulatory anti-

CTLA4 antibody, preferably, CP-675,206, and another therapeutic agent as part of a specific treatment regimen optionally including a maintenance phase, intended to provide a beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). "Combination therapy" generally is not intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.

"Combination therapy" embraces administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular, subcutaneous routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent (e.g., a chemotherapeutic agent) can be administered orally, and a second agent (e.g., anti- CTLA4 antibody) can be administered intravenously. Further, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, both the therapeutic agents may be administered by intravenous or subcutaneous injection.

In the present specification the term "sequential" means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of an anti-CTLA4 antibody and a chemotherapeutic agent, a sequential dosage regimen could include administration of the anti-CTLA4 antibody before, simultaneously, substantially simultaneously, or after administration of the chemotherapeutic agent, but both agents will be administered in a regular sequence or order. The term "separate" means, unless otherwise specified, to keep apart one from the other. The term "simultaneously" means, unless otherwise specified, happening or done at the same time, i.e., the compounds of the invention are administered at the same time. The term "substantially simultaneously" means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two compounds separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. Sequential administration refers to temporally separated administration of the anti-CTLA4 antibody and the chemotherapeutic agent.

"Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent, a dendritic cell vaccine or other tumor vaccine) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

As used herein, the term "adjuvant therapy" refers to treatment given after the primary treatment, including, without limitation, radiation, chemotherapy, hormone therapy, etc. The goal of adjuvant therapy is to increase the patients' chances of remission or cure, to increase the patients' overall survival benefit, and to help decrease the risk of recurrence. Therefore, it will be understood that if the anti-CTLA4 antibody and therapeutic agent combination is administered as adjuvant therapy, the combination may be administered to the patient after the primary treatment, e.g., the patient is given a regimen of surgery, radiation and/or chemotherapy, followed by a course of a combination of an anti-CTLA4 antibody and a therapeutic agent. In this regard, the dose of anti-CTLA4 antibody and therapeutic agent may be considered a therapeutic dose or a maintenance dose, depending on the goals of the adjuvant therapy. The term "neoadjuvant therapy" refers to treatment given before the primary treatment, including, without limitation, surgery, radiation, chemotherapy, etc. In the neoadjuvant setting, the dose of anti-CTLA4 antibody and therapeutic agent is considered a therapeutic dose.

The term "first-line therapy" refers to the first type of therapy given for a condition or disease, or the first therapy of choice for the treatment of a particular type of cancer. It necessarily follows that the term "second-line therapy" therefore refers to the treatment given when the initial or first-line therapy is unsuccessful, and "third-line therapy" refers to a treatment or treatment regimen that is given when both the initial treatment and the subsequent treatment are unsuccessful.

I. Combination Therapy A. Cancer

CTLA4 antibodies described herein can be used to treat a wide variety of cancers. Without wishing to be bound by any particular theory, administration of a therapeutic agent, when administered with an immunoenhancing anti-CTLA4 antibody, may provide a synergistic effect. Without wishing to be bound by any theory of the invention, the agent that mediates an anti-tumor effect may mediate a decrease in tumor load, may mediate an increase of tumor antigens in the host antigen-presentation route, may decrease inflammation such that the antibody and/or other therapeutic agents may better penetrate the tumor, and/or may mediate a decrease in immune-suppressive tumor factors. Thus, the combination of an anti- CTLA4 antibody and at least one therapeutic agent may mediate a synergistic therapeutic effect thereby providing a benefit to a patient in need thereof which is greater than either compound alone.

Cancers that may be treated include, but are not limited to human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, erythroblastoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, a primary or secondary brain tumor, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, skin cancer, bone cancer, cancer of the small intestine, cancer of the anal region, cancer of the head or neck, gastrointestinal (gastric, colorectal, and duodenal) cancer, esophageal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, cutaneous or intraocular melanoma, carcinoma of the endometrium, carcinoma of the vagina, carcinoma of the fallopian tubes, carcinoma of the vulva, carcinoma of the cervix, neoplasms of the central nervous system (CNS), including primary or secondary CNS tumors, spinal axis tumors, primary CNS lymphoma, brain stem glioma, pituitary adenoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, stomach cancer, colon cancer, cancer of the rectum, renal cell carcinoma (RCC), hepatoma, bile duct carcinoma, gall bladder cancer, liver cancer, kidney cancer, gastro-esophageal cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, cancer of the penis, lung carcinoma, small cell lung carcinoma (SCLC), non-small cell lung cancer (NSCLC), thyroid cancer, cancer of the parathyroid gland, carcinoid tumors, cancer of the urethra, cancer of the ureter, cancer of the renal pelvis, cancer of the endocrine system, cancer of the adrenal gland, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma), bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, pituitary adenoma, adrenocortical cancer, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, tumors of the blood vessels (including benign and malignant tumors such as hemangioma, hemangiosarcoma, hemangioblastoma and lobular capillary hemangioma), acoustic neuroma, oligodendroglioma, meningioma, melanoma, cholangiocarcinoma, neuroblastoma, retinoblastoma, leukemias, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic leukemia, lymphocytic leukemia, chronic myeloid leukemia, chronic myelogenous leukemia, chronic myelocytic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, lymphocytic lymphomas, polycythemia vera, lymphoma, Hodgkin's disease lymphoma, non-Hodgkin's disease lymphoma, cutaneous T-cell lymphoma (CTCL), cutaneous B-cell lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, soft tissue sarcomas, gastrointestinal stromal tumors (GIST), glioblastomas, or a combination of one or more of the foregoing cancers.

In preferred embodiments, the cancers include non-Hodgkin's lymphoma, non-small cell lung cancer, colorectal carcinoma, chronic myeloid leukemia, chronic lymphocytic leukemia, pancreatic cancer, breast cancer and ovarian cancer.

Various preferred combination therapies are described herein for a variety of cancers; however, in certain embodiments described herein, the invention is not limited to these, or any other, combination therapies.

1. Non-Hodgkin's lymphoma (NHL) In one embodiment, the invention includes administering CP-675,206 to treat NHL, in combination with at least one therapeutic agent, preferably, rituximab (RITUXAN; Genentech, San Francisco, CA).

The combination can be administered to treat a patient afflicted with indolent NHL or aggressive NHL. In one embodiment, the NHL is indolent NHL, and the combination of CP- 675,206 and rituximab comprises first-line therapy. In a further embodiment, the combination therapy is administered as second-line therapy following cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and/or CHOP-R (cyclophosphamide, doxorubicin, vincristine, and prednisone with rituximab). In yet a further embodiment, the combination is followed by administration of rituximab as a single agent (SA) therapy. The combination of rituximab and CP-675,206 may increase and/or prolong an anti-tumor cell response thereby providing a therapeutic benefit to a patient suffering from NHL. In one embodiment, a combination of CP-675,206 and rituximab is administered as a second-line therapy to treat aggressive NHL refractory to CHOP-R. In another embodiment, where the treatment is second-line therapy for aggressive NHL refractory to CHOP-R, the method further comprises administering rituximab as a single agent therapy following administration of the CP-675,206/rituximab combination.

In another aspect, the combination of CP-675,206 and rituximab is administered to a patient as a second-line therapy following another treatment including, but not limited to, a chemotherapeutic therapy or a bone marrow transplant. In one aspect, the CP-675,206 rituximab combination is administered following high-dose bone marrow ablative chemotherapy and bone marrow transplant (BMT). In another aspect, the combination is administered as a first-line therapy prior to bone marrow ablation chemotherapy and BMT. Methods well-known in the art for assessing therapeutic strategies for treatment of NHL, which consider various known factors such as age and condition of the patient, stage of the disease, and the like, are available to determine when the combination therapy is indicated to effectively treat a patient afflicted with aggressive NHL. 2. Non-small cell lung cancer (NSCLC)

In one embodiment, the invention includes a method for treating NSCLC comprising administering a combination comprising CP-675,206 and a therapeutic agent to a patient in need of treatment. In one aspect, the agent is a non-immunesuppressive chemotherapeutic agent, such as, but not limited to, gefitinib (IRESSA). The combination of CP-675,206 and therapeutic agent may increase the immune response to the tumor due to, inter alia, increased release of tumor antigen associated with the cytotoxic effect of the chemotherapeutic agent. Thus, the skilled artisan, based upon the disclosure provided herein, would appreciate that while any therapeutic agent that mediates cell death and/or release of tumor antigen can be used with an anti-CTLA4 antibody to provide an increased immune response to the tumor and, thereby, providing a therapeutic benefit to the patient. Moreover, the skilled artisan would further understand once armed with the teachings provided herein, that the therapeutic effect can be further enhanced wherein the therapeutic agent does not decrease the immune enhancing effect of the antibody. While gefitinib is a chemotherapeutic agent known not to decrease the immune response in a patient, the invention is not limited to this, or any other, particular therapeutic agent. Rather, in an embodiment, the invention encompasses administration of CP-675,206 and a therapeutic agent that may mediate a therapeutic response to a NSCLC tumor.

In one embodiment of the invention, treatment comprises administration combination therapy comprising CP-675,206 and platinum-based chemotherapy. In one aspect, CP- 675,206 is administered to a NSCLC patient following a course of platinum-based therapy as an adjuvant therapy. That is, a patient that has been administered a course of platinum- based therapy and where the disease has responded or remained stable is administered CP- 675,206. In one aspect, the patient NSCLC is Stage MIb (with effusion) or Stage IV disease that has responded or remained stable after about six cycles of a platinum-containing regimen. In a further aspect, the patient is administered CP-675,206 at least about three weeks after the last dose of platinum-based chemotherapy. In yet another aspect, CP- 675,206 is administered within about six weeks after the last dose of platinum-based chemotherapy. In another aspect, CP-675,206 is administered at least about three weeks after but within six weeks of the last dose of platinum-based therapy. In one aspect of the invention, platinum-based chemotherapy comprises a platinum-based compound selected from such compounds as cisplatin, carboplatin (PARAPLATIN), eptaplatin, lobaplatin, nedaplatin, oxaliplatin (ELOXATIN, Sanofi), streptozocin, satraplatin (JM-126).

In another embodiment of the present invention, treatment for lung cancer comprises administration of bevacizumab (AVASTIN) and CP-675,206. In one aspect, the lung cancer comprises non-small cell and small cell lung cancer. In another aspect, the lung cancer comprises small cell lung cancer.

In another embodiment, the cancer is NSCLC and the treatment encompasses administration of CP-675,206 in combination with a taxane, where a taxane can include, but is not limited to, docetaxel (TAXOTERE), and paclitaxel (TAXOL), in further combination with gemcitabine (GEMZAR).

In another embodiment of the invention, the invention encompasses a method for treating NSCLC comprising administration of a CP-675,206, a taxane (e.g., docetaxel and paclitaxel), and a platinum compound. In one aspect, the method for treating NSCLC is a first line therapy comprising administering a combination of CP-675,206, a taxane and a platinum compound where the taxane is paclitaxel and the platin is carboplatin (carbo/paclitaxel). In another aspect, the combination of CP-675,206, paclitaxel and carboplatin is administered contemporaneously, or in sequence. In yet a further aspect, the paclitaxel/carboplatin combination is administered prior to or following administration of CP-675,206. That is, the antibody is contemporaneously administered in combination with the carbo/paclitaxel therapy or it is administered following carbo/paclitaxel therapy. The interval between administration of carbo/paclitaxel therapy and administration of the antibody may be readily determined by one skilled in the art based upon well-known methods. In one aspect, the cancer is locally advanced Stage INb NSCLC. In another aspect, the NSCLC is metastatic Stage IV NSCLC. In another aspect, the CP-675,206-carboplatin-paclitaxel combination is administered with bevacizumab. In another aspect, the CP-675,206-carboplatin-paclitaxel combination is administered with sunitinib (SU11248). In a further aspect, the CP-675,206-carboplatin- paclitaxel combination is administered with PF03512676 (CpG-7909).

In another embodiment of the invention, the therapy is administration of docetaxel

(TAXOTERE) and CP-675,206 as second line therapy for NSCLC for patients with locally advanced Stage IHb or metastatic Stage IV disease after failure of prior platinum-based chemotherapy. In one aspect, CP-675,206 is administered contemporaneously with docetaxel therapy. In another aspect, CP-675,206 is administered following docetaxel therapy. In yet another aspect, the NSCLC comprises locally advanced Stage HIb or metastatic Stage IV non-small cell lung cancer after failure of prior platinum-based chemotherapy.

In yet another embodiment, the invention includes treatment for NSCLC where the therapy comprises administration of CP-675,206 and erlotinib (TARCEVA) as second line therapy. In one aspect, CP-675,206 is contemporaneously administered with erlotinib. In another aspect, CP-675,206 is administered following erlotinib therapy. In a further embodiment, the invention includes a second line therapy for NSCLC comprising administration of CP-675,206 and pemetrexed (ALIMTA). In one aspect, CP- 675,206 is administered contemporaneously with pemetrexed. In another aspect, CP- 675,206 is administered following pemetrexed therapy. In yet another aspect, the NSCLC is locally advanced Stage IHb. In yet a further aspect, the NSCLC is metastatic Stage IV non- small cell lung cancer after failure of prior platinum-based chemotherapy.

In another embodiment, the invention includes treatment of locally advanced Stage

IHb or metastatic Stage IV NSCLC after failure of prior platinum-based chemotherapy and epidermal growth factor receptor inhibition-based therapy where the method is a third line treatment. In one aspect, CP-675,206 is administered as a single agent. In another aspect, CP-675,206 is administered in combination with a therapeutic agent.

3. Colorectal carcinoma (CRC)

In one embodiment, the invention includes a method for treating cancer of the colon and/or rectum. In one embodiment, the method comprises administering a combination comprising CP-675,206 and fluorouracil (5FU) as first line therapy for CRC patients as first line therapy of CRC patients intolerant of oxaliplatin (ELOXATIN) or irinotecan (CAMPTO). In another embodiment, the treatment comprises administering a combination of CP-675,206 and capecitabine (XELODA) as first line therapy for patients intolerant of oxaliplatin or irinotecan.

In another embodiment, the therapy comprises first line treatment of patients with metastatic carcinoma of the colon or rectum comprising administration of CP-675,206 and

FOLFOX (fluorouracil, leucovorin, and oxaliplatin). In one aspect, CP-675,206 is administered contemporaneously with FOLFOX therapy. In another aspect, CP-675,206 is administered following FOLFOX therapy.

In another aspect, the treatment comprises adjuvant therapy of patients with Stage III colon cancer who have undergone complete resection of the primary tumor. In a further aspect , the therapy comprises administration of CP-675,206 and FOLFOX (fluorouracil, leucovorin, and oxaliplatin). In one aspect, CP-675,206 is administered contemporaneously with FOLFOX therapy. In another aspect, CP-675,206 is administered following FOLFOX therapy.

In another embodiment, the therapy comprises first line treatment of patients with metastatic CRC comprising administration of CP-675,206 and FOLFIRI (fluorouracil, leucovorin, and irinotecan). In one aspect, CP-675,206 is administered contemporaneously with FOLFIRI therapy. In another aspect, CP-675,206 is administered following FOLFIRI therapy.

In another embodiment of the invention, the therapy comprises treatment of CRC comprising administration of CP-675,206 and cetuximab (ERBITUX, ImClone). In one aspect, CP-675,206 is administered contemporaneously with cetuximab. In another aspect, CP- 675,206 is administered following cetuximab.

In another embodiment, the invention encompasses a method of treating CRC comprising administering a combination of CP-675,206 and fluorouracil and capecitabine as adjuvant therapy. Coadministration of a chemotherapeutic agent, which can mediate increased release of a tumor antigen, and CP-675,206 may mediate an increased immune response to the tumor thereby providing a therapeutic benefit to a patient afflicted therewith.

4. Leukemia

In one embodiment, the invention includes a method of treating chronic myeloid leukemia (CML). In one aspect, the method comprises administering a combination of CP- 675,206 and imatinib mesylate (GLEEVEC, Novartis) as first line therapy of CML.

In another embodiment, the invention includes a method for treating chronic lymphocytic leukemia (CLL). In one aspect, the treatment comprises administration of CP- 675,206 and imatinib mesylate as first line therapy. 5. Pancreatic cancer

In one embodiment, the invention includes a method for treating pancreatic cancer comprising administering a combination of CP-675,206 and gemcitabine (GEMZAR). In one aspect, the therapy is a first line treatment for patients with locally advanced (non-resectable

Stage Il or Stage III) adenocarcinoma of the pancreas. In another aspect, the cancer is metastatic (Stage IV) adenocarcinoma of the pancreas. In yet another aspect, gemcitabine is administered contemporaneously with CP-675,206. In yet a further aspect, CP-675,206 is administered following administration of gemcitabine.

In one embodiment of the invention, CP-675,206 is administered after at least one course of gemcitabine. In one aspect, gemcitabine is administered intravenously at a dose of about 1000 mg/m2 once per week for up to seven (7) weeks or until toxicity necessitates reducing or withholding a dose. The administration phase is followed by a one week resting period during which gemcitabine is not administered. Subsequent cycles of gemcitabine administration consist of i.v. infusions once weekly for three consecutive weeks followed by one week resting period. In a further aspect, CP-675,206 is administered after a full course (Ae., seven weeks administration and one week resting period) of gemcitabine. In another embodiment, CP-675,206 is administered every three weeks thereafter. In yet another aspect, the combination of gemcitabine (e.g., three weeks administration followed by one week rest) and CP-675,206 (e.g., every three weeks) is continued until disease progression or intolerable toxicity. 6. Breast cancer

In one embodiment, the invention includes a method to treat locally advanced or metastatic triple receptor negative breast cancer. In one aspect, the method is a first line therapy. In another aspect, the therapy comprises administering CP-675,206 and a taxane (e.g., docetaxel and paclitaxel). In one aspect, CP-675,206 taxane are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of the taxane.

In another embodiment, the therapy comprises adjuvant treatment for breast cancer comprising administration of CP-675,206 and cyclophosphamide, doxorubicin and a taxane. In one aspect, the therapy comprises adjuvant therapy. In another embodiment, therapy comprises administration of CP-675,206 and tamoxifen (NOVALDEX), anastrazole (ARIMIDEX), letrozole (FEMARA), exemestane (AROMASIN), or fulvestrant (FASLODEX), or a combination thereof. In one aspect, the therapy is adjuvant therapy for metastatic breast cancer.

In another embodiment of the present invention, the therapy for breast cancer comprises administration of CP-675,206 and trastuzumab (HERCEPTIN). In one aspect, the antibodies are administered contemporaneously. In another aspect, trastuzumab is administered followed by administration of CP-675,206. In a further aspect, the CP-675,206- trastuzumab combination therapy comprises adjuvant therapy and first line therapy for metastatic breast cancer. In yet another embodiment, the therapy comprises administering CP-675,206 and bevacizumab. In one aspect, CP-675,206 and bevacizumab are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of bevacizumab.

In one embodiment of the invention, the therapy for treating breast cancer comprises administering CP-675,206 and cetuximab. In one aspect, CP-675,206 and cetuximab are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of cetuximab.

In another embodiment of the invention, the therapy for treating breast cancer comprises administering CP-675,206 and axitinib. In one aspect, CP-675,206 and axitinib are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of axitinib.

7. Ovarian cancer

In one embodiment, the invention includes a method for treatment carcinoma of the ovary. In one embodiment, therapy comprises administration of CP-675,206 and carboplatin and paclitaxel. In one aspect, CP-675,206 is administered contemporaneously with carboplatin and paclitaxel. In another aspect, CP-675,206 is administered following administration of carboplatin and paclitaxel. In yet another aspect, the treatment is a first line treatment for advanced carcinoma of the ovary.

In another embodiment of the invention, therapy comprises second line treatment for patients who have progressed (e.g., as indicated by tumor assessment, CA-125 doubling value, CT scan, and the like) following paclitaxel-based therapy. In one aspect, therapy comprises administration of CP-675,206 as a single agent. In another aspect, therapy comprises administration of CP-675,206 in combination with an agent selected from the group consisting of altretamine, anastrozole, bevacizumab, carboplatin, cisplatin, cyclophosphamide, liposomal doxorubicin, docetaxel, gemcitabine, ifosfamide, irinotecan, letrozole, melphalan, oral etoposide, oxaliplatin, tamoxifen, topotecan, and vinorelbine, and any combination thereof.

8. Gastric cancer

In one embodiment of the present invention, the treatment comprises gastric cancer and therapy comprises administering CP-675,206 and irinotecan. In one aspect, CP-675,206 and irinotecan are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of irinotecan.

In another embodiment, treatment comprises administration of CP-675,206, fluorouracil and leucovorin. In one aspect, CP-675,206, fluorouracil and leucovorin are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of fluorouracil and leucovorin.

9. Liver cancer In one embodiment of the invention, the cancer is liver cancer and therapy comprises administering CP-675,206 and doxorubicin, ifosfamide and vincristine. In one aspect, CP-

675,206, and doxorubicin, ifosfamide and vincristine are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of doxorubicin, ifosfamide and vincristine.

In another embodiment, the cancer is liver cancer and therapy comprises administering CP-675,206, doxorubicin, and vincristine. In one aspect, CP-675,206, and doxorubicin, ifosfamide and vincristine are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of doxorubicin and vincristine. 10. Metastatic lesions

In one embodiment of the present invention, CP-675,206 is administered to treat metastases where the metastases is to the brain and/or bone metastasis. In one aspect, the brain and bone metastases are treated using CP-675,206 regardless of the site of the primary tumor from which the cancer metastasized. In one aspect, brain metastases are treated using a combination of surgical resection and/or radiotherapeutic ablation in further combination with an anti-CTLA4 antibody, preferably, CP-675,206. That is, where multiple metastases and/or lesions greater than about 3 centimeters in diameter are present, whole brain radiation may be administered followed by administration of anti-CTLA4 antibody. In a further aspect, the antibody is administered preceding radiation. In yet a further aspect, the antibody is administered at least about 3 mg/kg, more preferably, at least about 6 mg/kg, even more preferably, at least about 10 mg/kg, and more preferably, at least about 15 mg/kg. In another aspect, the antibody is administered every three weeks, more preferably, every four weeks, and even more preferably, every three months. In one embodiment, where there are fewer than about 5 brain lesions and all lesions are less than about three centimeres in maximal diameter, stereotactic radiosurgery (SRS) is administered followed by administration of anti-CTLA4 antibody. In one aspect, the antibody is administered before SRS. In another aspect, the antibody is administered before and after SRS. In another embodiment, surgery, whole brain radiation and SRS may be combined in any order to treat brain metastases, which treatment is further combined with antibody therapy. In one aspect, the antibody therapy is administered after radiation/surgery/SRS. Alternatively, antibody surgery is administered before radiation/surgery/SRS and may be administered before and after such therapy. This is because, as would be understood by the skilled artisan, brain metastases are common in melanoma. While SRS may achieve local control of the brain metastases, most patients die of systemic metastases and/or reseeding of the CNS occurs due to poor management of the systemic disease. Accordingly, in one embodiment, the invention provides a combination therapy to control the local brain metastases and to prevent reseeding of the CNS and control the systemic disease thereby providing a therapeutic benefit to a patient in need thereof.

11. Bladder cancer

In one embodiment, CP-675,206 is administered in combination with BCG to treat bladder cancer. In one aspect, CP-675,206 and BCG are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of BCG. In another embodiment, treatment of bladder cancer comprises administering CP-

675,206 and gemcitabine and cisplatin. In one aspect, CP-675,206, gemcitabine and cisplatin are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of gemcitabine and cisplatin.

12. Melanoma The present invention encompasses combination of CP-675,206 and interferon alpha for treating melanoma. In one aspect, CP-675,206 and interferon alpha are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of interferon alpha. In a further aspect, interferon alpha is administered as a high dose.

13. Multiple myeloma In one embodiment of the present invention, treatment for multiple myeloma comprises administration of CP-675,206 and bortezomib. In one aspect, CP-675,206 and bortezomib are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of bortezomib.

In another embodiment, therapy comprises administration of CP-675,206 and dexamethasone and thalidomide. In one aspect, CP-675,206, dexamethasone and thalidomide are administered contemporaneously. In another aspect, CP-675,206 is administered following administration of dexamethasone and thalidomide.

In alternate embodiments of the invention discussed above, anti-CTLA4 antibodies other than CP-675,206 may be used in the methods of the invention. Alternate antibodies are described herein, including the antibodies described in U.S. Patent Application No. 09/472,087, now issued as U.S. Patent No. 6,682,736; Int. Appl. No. PCT/US99/30895 (published June 29, 2000, as WO 00/37504); U.S. Pat. Appl. No. 10/612,497 (published November 18, 2004, as US 2004/0228858); U.S. Pat. Appl. No. 10/776,649 (published November 18, 2004, as US 2004/0228861); Int. Appl. No. Int. Appl. No. PCT/USOO/23356 (published March 1 , 2001, as WO 01/14424) (e.g., antibody 10D1 , also known as MDX-010, and referred to herein as ipilimumab, Medarex, Princeton, NJ); Int. Appl. No. PCT/US99/28739 (published June 8, 2000, as WO 00/32231); U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051 ,227, and 6,207,156; U.S. Pat. No. 5,844,095, to Linsley et al.; Int. Appl. No. PCT/US92/05202 (published Jan. 7, 1993, as WO 93/00431); U.S. Patent Appl. No. 10/153,382 (published May 8, 2003, as US 2003/0086930); U.S. Pat. Appl. No. 10/673,738 (published February 24, 2005 as US 2005/0042223); U.S. Pat. Appl. No. 11/085,368 (published October 13, 2005, as US 2005/0226875); U.S. Pat. Appl. No. 60/624,856 (filed Nov. 4, 2004); U.S. Pat. Appl. No. 60/664,364 (filed March 23, 2005); U.S. Pat. Appl. No. 60/664,653 (filed March 23, 2005); U.S. Pat. Appl. No. 60/697,082 (filed July 7, 2005); U.S. Pat. Appl. No. 60/711,707 (filed Aug. 26, 2005). More preferably, the antibodies are described in U.S. Patent No. 6,682,736, and WO 01/14424. Even more preferably, the antibodies include an antibody having the heavy and light chain amino acid sequences of an antibody selected from the group consisting of 4.1.1 , 4.8.1 , 4.10.2, 4.13.1, 4.14.3, 6.1.1 , 11.2.1 (CP-675,206 or ticilimumab), 11.6.1 , 11.7.1., 12.3.1.1, 12.9.1.1, and ipilimumab. B. Pathogens Because of the immune-enhancing effects of CTLA4-blockade, it would be understood by the skilled artisan armed with the disclosure provided herein that the present invention encompasses administration of anti-CTLA4 antibody in combination with standard of care therapy to treat a variety of pathogen-associated diseases, disorders or conditions, where an immune response to the pathogen would provide a therapeutic benefit. These include, but are not limited to, bacterial, viral, fungal, parasitic, and other pathogenic disease. Treatment of infectious disease comprising anti-CTLA4 antibody blockade is discussed in, e.g., WO 03/086459, published on Oct. 23, 2003.

Similar to use of CTLA4 blockade in combination to treat a tumor discussed previously, anti-CTLA4 antibody blockade can be used in combination with vaccines and other therapeutic agents, including standard of care therapy, to treat a disease, disorder or condition associated with a pathogens, or a toxin derived therefrom. That is, previous studies have demonstrated that anti-CTLA4 antibody blockade can be useful in treatment of infectious parasites (McCoy et al., J. Exp. Med. 186:183-187 (1997); Murphy et al., J. Immunol. 161 :4153-4160 (1998)). Examples of pathogens for which this therapeutic approach may be particularly useful, include, but are not limited to HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aureginosa, and Helicobacter pylori.

Some examples of viruses treatable by methods of the invention include hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1 , HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, ebola virus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, poxvirus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

In one embodiment, bacterial infection treatable by methods of the invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, helicobacter, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme Disease bacteria (e.g., Borrelia burgdorferi).

In another embodiment, fungal infection treatable according to the methods of the invention include, but are not limited to, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

In yet another embodiment, parasitic infection treatable by methods of the invention include, among others, Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylus brasiliensis. 1. HIV/AIDS Studies suggest that CTLA4 blockade may be useful for treatment of viral disease, more specifically, studies in rhesus macaques demonstrated that anti-CTLA4 (i.e., ipilimumab), combined with antiretroviral therapy ("ART"), was associated with increased antiviral response. See Hryniewicz et al., Blood 108:3834-3842 (2006). More particularly, CTLA4 blockade not only decreased viral load and increased virus-specific effector T cells, it also decreased the level of immunosuppressive molecules (e.g., transforming growth factor beta [TGF-β] and indoleamine 2,3-dioxygenase [IDO]), in SIVmac25i-infected macaques also receiving ART (didanosine, stavudine and tenofovir [PMPA]). These data suggest that anti- CTLA4 antibody, in combination with antiviral therapy, may provide a therapeutic benefit in treatment of HIV infection in humans. In one embodiment of the invention, anti-CTLA4 antibodies, in particular CP-675,206, and their pharmaceutically acceptable salts, solvates and derivatives, may be administered alone or as part of a combination therapy. Thus included within the scope of the present invention are embodiments comprising co-administration of, and compositions which contain, in addition to a compound of the invention, one or more additional therapeutic agents. Such multiple drug regimens, often referred to as combination therapy, may be used in the treatment and prevention of infection and multiplication of the human immunodeficiency virus, HIV, and related pathogenic retroviruses within a patient in need of treatment or one at risk of becoming such a patient. The ability of such retroviral pathogens to evolve within a relatively short period of time into strains resistant to any monotherapy which has been administered to said patient is well known in the literature. A recommended treatment for HIV is a combination drug treatment called Highly Active Anti-Retroviral Therapy ("HAART"). HAART combines three or more HIV drugs. Thus, the methods of treatment and pharmaceutical compositions of the present invention may employ a compound of the invention in the form of monotherapy, but said methods and compositions may also be used in the form of combination therapy in which anti-CTLA4 antibody is co-administered in combination with one or more additional therapeutic agents such as those described in detail further herein.

The therapeutic agents that may be used in combination with the anti-CTLA4 antibody include, but are not limited to, those useful as HIV protease inhibitors (PIs), non- nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), CCR5 antagonists, agents which inhibit the interaction of gp120 with CD4, other agents which inhibit the entry of HIV into a target cell (such as fusion inhibitors), inhibitors of HIV integrase, RNaseH inhibitors, prenylation inhibitors, maturation inhibitors which act by interfering with production of the HIV capsid protein, compounds useful as anti-infectives, and others as described below. It will be appreciated by a person skilled in the art, that a combination drug treatment, as described herein above, may comprise two or more compounds having the same, or different, mechanism of action. Thus, by way of illustration only, a combination may comprise a compound of the invention and: one or more NRTIs; one or more NRTIs and a Pl; one or more NRTIs and another CCR5 antagonist; a Pl; a Pl and an NNRTI; an NNRTI; and so on. Examples of PIs include, but are not limited to, amprenavir (141W94), CGP-73547,

CGP-61755, DMP-450 (mozenavir), nelfinavir, ritonavir, saquinavir (invirase), lopinavir, TMC- 126, atazanavir, palinavir, GS-3333, KN 1-413, KNI-272, LG-71350, CGP-61755, PD 173606, PD 177298, PD 178390, PD 178392, U-140690, ABT-378, DMP-450, AG-1776, MK-944, becanavir (formerly known as VX-478, GW640385), indinavir, tipranavir, TMC-114, DPC-681, DPC-684, fosamprenavir calcium (Lexiva), benzenesulfonamide derivatives disclosed in WO 03/053435, R-944, Ro-03-34649, VX-385, GS-224338, OPT-TL3, PL-100, PPL-100, SM- 309515, AG-148, DG-35-VIII, DMP-850, GW-5950X, KNI-1039, L-756423, LB-71262, LP-130, RS-344, SE-063, UIC-94-003, Vb-19038, A-77003, BMS-182193, BMS-186318, SM-309515, JE-2147, GS-9005. Examples of NRTIs include, but are not limited to, abacavir, GS-840, lamivudine, adefovir dipivoxil, beta-fluoro-ddA, zalcitabine, didanosine, stavudine, zidovudine, tenofovir (9-[9(R)-2-(phosphonomethoxy)propyl]adenine; PMPA), tenofovir disoproxil fumarate (Viread; Gliead Sciences), amdoxovir (DAPD), SPD-754, SPD-756, racivir, reverset (DPC-817), MIV- 210 (FLG), beta-L-Fd4C (ACH-126443), MIV-310 (alovudine, FLT), dOTC, DAPD, entecavir, GS-7340, emtricitabine (FTC). Examples of NNRTIs include, but are not limited to, efavirenz, HBY-097, nevirapine,

TMC-120 (dapivirine), TMC-125, etravirine, delavirdine, DPC-083, DPC-961 , capravirine, rilpivirine, 5-{[3,5-Diethyl-1 -(2-hydroxyethyl)-1 A7-pyrazol-4-yl]oxy}isophthalonitrile or pharmaceutically acceptable salts, solvates or derivatives thereof; GW-678248, GW-695634, MIV-150, calanolide, and tricyclic pyrimidinone derivatives as disclosed in WO 03/062238. Examples of CCR5 antagonists include, but are not limited to, TAK-779, SC-351125, ancriviroc (formerly known as SCH-C), vicriviroc (formerly known as SCH-D), maraviroc, NCB-9471, CCR5mAb004, PRO-140, aplaviroc (also known as GW-873140, Ono-4128, AK- 602), AMD-887 CMPD-167, methyl 1-eA7do-{8-[(3S)-3-(acetylamino)-3-(3-fluorophenyl)propyl]- S-azabicyclop^.^oct-S-ylϊ^-methyl^.δ.θJ-tetrahydro-IW-imidazoμ.δ-clpyridine-δ- carboxylate or pharmaceutically acceptable salts, solvates or derivatives thereof, methyl 3- enc/o-{8-[(3S)-3-(acetamido)-3-(3-fluorophenyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-2-methyl- 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-5-carboxylate or pharmaceutically acceptable salts, solvates or derivatives thereof, ethyl 1-endo-{8-[(3S)-3-(acetylamino)-3-(3- fluorophenyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-2-methyl-4,5,6,7-tetrahydro-1/-/-imidazo[4,5- c]pyridine-5-carboxylate or pharmaceutically acceptable salts, solvates or derivatives thereof, and Λ/-{(1 S)-3-[3-enαfo-(5-lsobutyryl-2-methyl-4,5,6,7-tetrahydro-1 H-imidazo[4,5-c]pyridin-1 - yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-(3-fluorophenyl)propyl}acetamide) or pharmaceutically acceptable salts, solvates or derivatives thereof.

In one embodiment, the anti-CTLA4 antibody, preferably CP-675,206, is administered in combination with maraviroc. In another embodiment, anti-CTLA4 antibody/maraviroc combination therapy comprises assessing whether is expressed on the surface of the patient's cells. In yet another embodiment, the therapy comprises assessing the co-receptor {i.e., CXCR4 or CCR5) tropism of the HIV virus to determine whether the patient is infected with an HIV that uses the CCR5 co-receptor, the CXCR4 co-receptor, or both (dual/mixed tropic) to determined whether the patient is a candidate for maraviroc therapy. In another embodiment, the co-receptor tropism assay is TROFILE (Monogram Biosciences, Inc., San Francisco, CA). This is because treatment with maraviroc inhibits CCR5-mediated entry into cells. Thus, assessing CCR5 expression by a patient's cell and/or assessing the co-receptor tropism of the virus optimizes the treatment options for the patient. Examples of entry and fusion inhibitors include, but are not limited to, BMS-806,

BMS-488043, 5-{(1S)-2-[(2R)-4-Benzoyl-2-methyl-piperazin-1-yl]-1-methyl-2-oxo-ethoxy}-4- methoxy-pyridine-2-carboxylic acid methylamide and 4-{(1S)-2-[(2R)-4-Benzoyl-2-methyl- piperazin-1-yl]-1-methyl-2-oxo-ethoxy}-3-methoxy-N-methyl-benzamide, enfuvirtide (T-20), sifuvirtide SP-01A, T1249, PRO 542, AMD-3100, soluble CD4, compounds disclosed in JP 2003171381 , and compounds disclosed in JP 2003119137. Examples of inhibitors of HIV integrase include, but are not limited to, L-000870810

GW-810781, 1 ,5-naphthyridine-3-carboxamide derivatives disclosed in WO 03/062204, compounds disclosed in WO 03/047564, compounds disclosed in WO 03/049690, and 5- hydroxypyrimidine-4-carboxamide derivatives disclosed in WO 03/035076, MK-0518 (5-(1 ,1- dioxo-1 ,2-thiazinan-2-y))-N- (4-fluorobenzyl)-8-hydroxy-1 ,6-naphthyridine-7-carboxamide- disclosed in WO 03016315), GS-9137 (JTK-303).

Examples of prenylation inhibitors include, but are not limited to, HMG CoA reductase inhibitors, such as statins (e.g., atorvastatin).

Examples of maturation inhibitors include 3-O-(3'3'-dimethylsuccinyl) betulic acid (otherwise known as PA-457) and alphaHGA.

II. Dosage Regimens

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non limiting range for a therapeutically effective amount of ticilimumab administered according to the invention is at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, more than about 10 mg/kg, or at least about 15 mg/kg, for example about 1-30 mg/kg, or for example about 1-25 mg/kg, or for example about 1-20 mg/kg, or for example about 5-20 mg/kg, or for example about 10-20 mg/kg, or for example about 15-20 mg/kg, or for example, about 15 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Determining appropriate dosages and regiments for administration of the antibody are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

In one embodiment, CP-675,206 is administered in an intravenous formulation as a sterile aqueous solution containing about 5 to 20 mg/ml of antibody, in an appropriate buffer system. In one embodiment, for administration of low doses, part of the dose is administered by an intravenous bolus and the rest by infusion of the antibody formulation. For example, a 0.01 mg/kg intravenous injection of the antibody may be given as a bolus, and the rest of a predetermined antibody dose may be administered by intravenous injection. In another embodiment, the entire low dose is administered as a single bolus injection. For higher doses, e.g., 3 mg/kg, the antibody is not administered as a bolus, but the entire amount is administered by infusion. A predetermined dose of the antibody may be administered, for example, over a period of about an hour and a half to about five hours.

The present invention relates to administering a combination of an anti-CTLA4 antibody and at least one therapeutic agent. In one embodiment, the antibody is CP-675,206. In alternative embodiments of the invention, the antibody is selected from various anti-CTLA4 antibodies, including, but not limited to, ipilimumab. In one aspect, the antibody is ipilimumab and the dose is about 3 mg/kg. In another aspect, the dose of ipilimumab is at least about 10 mg/kg.

In one embodiment of the present invention, the antibody (e.g., CP-675,206, ipilimumab, and the like) is administered approximately every three weeks, more preferably, for about four cycles followed by every three months thereafter. In one aspect of this embodiment, the antibody is administered at about 10 mg/kg.

The skilled artisan would appreciate that the combination of CP-675,206 and a therapeutic agent can be administered simultaneously or the antibody and the therapeutic agent can be administered at different times. For instance, in one embodiment, the antibody is administered as a single injection and/or infusion and the therapeutic agent (e.g., gemcitabine) is administered once per day commencing before, during, or after administration of the antibody. However, the present invention is not limited to any particular dosage or administration regimen for a therapeutic agent. Rather, the optimal dose, route and regimen for administration of the antibody and the therapeutic agent can be readily determined by one of ordinary skill in the relevant art using well-known methods. For instance, a single dose or multiples doses of the antibody may be administered. Alternatively, at least one dose, or at least three, six or 12 doses may be administered. The doses may be administered, for example, every two weeks, every three weeks, monthly, every twenty days, every 25 days, every 28 days, every 30 days, every 40 days, every 6 weeks, every 50 days, every two months, every 70 days, every 80 days, every three months, every six months or yearly. In one aspect, the antibody is administered once every three weeks, preferably for four cycles, and then every three months thereafter. In addition, the second therapeutic agent can be administered daily, several times or once per day, weekly, every other week, every third week, every fourth week, monthly, every three months, every six months, once per year, or any other period that provides a therapeutic benefit to the patient as determined by the skilled practitioner.

In one aspect, CP-675,206 is administered once per month. In another aspect, CP- 675,206 is administered every three months. In yet another aspect, ipilimumab is administered once per month. The antibody can be administered until disease progression, or intolerable toxicity, or up to 12 consecutive cycles, whichever time is shorter. The antibody can also be administered using a regimen comprising administration of a loading dose followed by a lower dose. Repeat courses of at least one, and more preferably, several cycles of antibody and a therapeutic agent can be administered to a patient that experiences a tumor recurrence who had previously derived benefit from administration of the combination of an anti-CTLA4 antibody and another therapeutic agent, or the antibody or therapeutic agent not administered in combination previously.

In one embodiment, a single injection comprising CP-675,206 is administered to a patient intravenously at a dose of about 3 mg/kg every twenty-eight days. In another embodiment, an anti-CTLA4 antibody is administered to a patient intravenously at a dose of about 3 mg/kg, preferably, about 6 mg/kg, more preferably, about 10 mg/kg, preferably, about 15 mg/kg, every three weeks. After four administrations, the antibody is then administered every three months.

The invention encompasses administration of an anti-CTLA4 antibody, preferably CP- 675,206, in combination with a wide plethora of agents. The skilled artisan would appreciate, once armed with the teachings provided herein, that any art-recognized dosing regimen for the agent may be used. Dosing regimens for chemotherapeutic and other agents described herein may be found in treatises well known in the art, including, but not limited to, Cancer: Principles and Practice of Oncology, 7th edition, DeVita, Hellman, and Rosenberg, editors, Lippincott, Williams & Wilkins, 2004, and Cancer Chemotherapy and Biotherapy Principles and Practice, 4th edition. Chabner, Longo, editors, Lippincott, Williams & Wilkins, 2005. Additionally, administration of many agents disclosed herein may be performed based upon the FDA- approved drug label instructions. Such label instructions are readily and publicly available from the website of the Department of Health and Sciences, U.S. Food and Drug Administration, Center for Drug Evaluation and Research website, which may be searched according to drug name or active ingredient. Thus, the skilled artisan would understand the dosing regimen to be used in combination therapy for various diseases comprising administration of an anti-CTLA4 antibody and a therapeutic agent. The dose may be adjusted as known in the art based on, among other factors, toxicity, if any, and therapeutic effectiveness. Thus, dosing regimens for many chemotherapeutic, and other agents related to cancer treatment, are well known in the art and are described in numerous publications, including those described herein. See also International Publication No. PCT/US03/12802 (published Nov. 6, 2003, as WO 03/090686), which is incorporated by reference herein for all purposes, for a list of exemplary dosing regimens for various chemotherapeutic agents (e.g., at pages 72 through 74).

In some embodiments, the antibody-therapeutic agent combination can be administered as an adjuvant therapy, or it can be administered to the patient as a neoadjuvant therapy prior to surgery, radiation therapy, or any other treatment, in order to sensitize the tumor cells or to otherwise confer a therapeutic benefit to the patient.

Further, in some embodiments, the combination can be administered as a first line therapy, or as a second line or third line therapy, such as, but not limited to, once previous therapy(ies) has failed. Alternatively, the combination can be administered concurrently with a first line therapy, and or at any point during therapy, following initial treatment. Thus, a combination of an anti-CTLA4 antibody and a therapeutic agent can provide a therapeutic benefit once a previous therapy has failed, once systemic adjuvant therapy using another therapeutic agent (e.g., temozolomide, leuprolide, paclitaxel, imatinib mesylate, gefitinib, and the like) has failed. Therefore, the invention encompasses administration of an anti-CTLA4 antibody and a therapeutic agent in combination with or following additional therapy, including, but not limited to, a different therapeutic agent (e.g., a chemotherapeutic, an antibody, an immunomodulator, a cytokine, and the like) as would be appreciated by one skilled in the art based upon the disclosure provided herein.

III. Anti-CTLA4 Antibodies

The invention encompasses alternate embodiments employing CTLA4 antibodies in addition to CP-675,206. In one embodiment, the CTLA4 antibody comprises a heavy chain wherein the amino acid sequence of the VH comprises the amino acid sequences set forth in SEQ ID NOs:3, 15 and 27. In yet another embodiment, the VL of the CTLA4 antibody comprises the amino acid sequences set forth in SEQ ID NOs:9, 21 and 33. More preferably, the VH and VL regions of the antibody comprise the amino acid sequences set forth in SEQ ID NO:3 (VH 4.1.1) and SEQ ID NO:9 (VL 4.1.1), respectively; the amino acid sequences set forth in SEQ ID NO:15 (VH 4.13.1) and SEQ ID NO:21 (VL 4.13.1 ), respectively; and the amino acid sequences set forth in SEQ ID NO:27 (VH 11.2.1) and SEQ ID NO:33 (VL 11.2.1), respectively. Most preferably, the antibody is CP-675,206, which has the heavy and light chain amino acid sequences of antibody 11.2.1 (ticilimumab).

In yet another embodiment, the amino acid sequence of the heavy chain comprises the amino acid sequence encoded by a nucleic acid comprising the nucleic acid sequences set forth in SEQ ID NOs:1, 13, and 25. In yet another embodiment, the light chain comprises the amino acid sequence encoded by a nucleic acid comprising the nucleic acid sequences set forth in SEQ ID NOs:7, 19 and 31. More preferably, the heavy and light chains comprise the amino acid sequences encoded by nucleic acids comprising the nucleic acid sequences set forth in SEQ ID NO:1 (heavy chain 4.1.1) and SEQ ID NO:7 (light chain 4.1.1), respectively; the nucleic acid sequences set forth in SEQ ID NO: 13 (heavy chain 4.13.1) and SEQ ID NO:19 (light chain 4.13.1), respectively; and the nucleic acid sequences set forth in SEQ ID NO:25 (heavy chain 11.2.1) and SEQ ID NO:31 (light chain 11.2.1), respectively.

Furthermore, the antibody can comprise a heavy chain amino acid sequence comprising human CDR amino acid sequences derived from the VH 3-30 or 3-33 gene, or conservative substitutions or somatic mutations therein. It is understood that the VH 3-33 gene encodes from FR1 through FR3 of the heavy chain variable region of an antibody molecule. Thus, the invention encompasses an antibody that shares at least 85%, more preferably, at least 90%, yet more preferably, at least 91 %, even more preferably, at least 94%, yet more preferably, at least 95%, more preferably, at least 97%, even more preferably, at least 98%, yet more preferably, at least 99%, and most preferably, 100% identity, with the sequence from FR1 through FR3 of an antibody selected from the group consisting of 3.1.1, 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP-675,206), 11.6.1 , 11.7.1 , 12.3.1.1 , 2.9.1.1 , ipilimumab, and DP-50.

The antibody can further comprise CDR regions in its light chain derived from the A27 or the 012 gene or it may comprise the CDR regions of an antibody selected from the group consisting of 3.1.1, 4.1.1 , 4.8.1, 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP-675,206), 11.6.1, 11.7.1, 12.3.1.1 , 2.9.1.1 , ipilimumab.

In other embodiments of the invention, the antibody inhibits binding between CTLA4 and B7-1 , B7-2, or both. Preferably, the antibody can inhibit binding with B7-1 with an IC50 of about 100 nM or lower, more preferably, about 10 nM or lower, for example about 5 nM or lower, yet more preferably, about 2 nM or lower, or even more preferably, for example, about 1 nM or lower. Likewise, the antibody can inhibit binding with B7-2 with an IC50 of about 100 nM or lower, more preferably, 10 nM or lower, for example, even more preferably, about 5 nM or lower, yet more preferably, about 2 nM or lower, or even more preferably, about 1 nM or lower.

Further, in another embodiment, the anti-CTLA4 antibody has a binding affinity for CTLA4 of about 10"8, or greater affinity, more preferably, about 10"9 or greater affinity, more preferably, about 10~10 or greater affinity, and even more preferably, about 10"11 or greater affinity.

The anti-CTLA4 antibody includes an antibody that competes for binding with an antibody having heavy and light chain amino acid sequences of an antibody selected from the group consisting of 4.1.1 , 6.1.1 , 11.2.1 (CP-675,206), 4.13.1 and 4.14.3. Further, the anti- CTLA4 antibody can compete for binding with antibody ipilimumab.

In another embodiment, the antibody preferably cross-competes with an antibody having a heavy and light chain sequence, a variable heavy and a variable light chain sequence, and/or the heavy and light CDR sequences of antibody 4.1.1 , 4.13.1 , 4.14.3, 6.1.1. or 11.2.1 (CP-675,206). For example, the antibody can bind to the epitope to which an antibody that has heavy and light chain amino acid sequences, variable sequences and/or CDR sequences, of an antibody selected from the group consisting of 4.1.1 , 4.13.1, 4.14.3, 6.1.1 , or 11.2.1 (CP-675,206) binds. In another embodiment, the antibody cross-competes with an antibody having heavy and light chain sequences, or antigen-binding sequences, of MDX-D010.

In another embodiment, the invention is practiced using an anti-CTLA4 antibody that comprises a heavy chain comprising the amino acid sequences of CDR-1 , CDR-2, and CDR- 3, and a light chain comprising the amino acid sequences of CDR-1 , CDR-2, and CDR-3, of an antibody selected from the group consisting of 3.1.1 , 4.1.1 , 4.8.1, 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP-675,206), 11.6.1 , 11.7.1, 12.3.1.1 , and 12.9.1.1 , or sequences having changes from the CDR sequences selected from the group consisting of conservative changes, wherein the conservative changes are selected from the group consisting of replacement of nonpolar residues by other nonpolar residues, replacement of polar charged residues other polar uncharged residues, replacement of polar charged residues by other polar charged residues, and substitution of structurally similar residues; non-conservative substitutions, wherein the non-conservative substitutions are selected from the group consisting of substitution of polar charged residue for polar uncharged residues and substitution of nonpolar residues for polar residues, additions and deletions.

In a further embodiment of the invention, the antibody contains fewer than 10, 7, 5, or 3 amino acid changes from the germline sequence in the framework or CDR regions. In another embodiment, the antibody contains fewer than 5 amino acid changes in the framework regions and fewer than 10 changes in the CDR regions. In one preferred embodiment, the antibody contains fewer than 3 amino acid changes in the framework regions and fewer than 7 changes in the CDR regions. In a preferred embodiment, the changes in the framework regions are conservative and those in the CDR regions are somatic mutations.

In another embodiment, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 94%, preferably, at least 95%, more preferably, at least 99%, sequence(e.g., amino acid, nucleic acid, or both) identity or sequence similarity over the heavy and light chain full-length sequences, or over the heavy or the light chain, separately, with the sequences of antibody 3.1.1 , 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1, 11.2.1 (CP-675,206), 11.6.1 , 11.7.1 , 12.3.1.1 , 12.9.1.1 , ipilimumab. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy chain and the light chain, or with the heavy chain or the light chain, separately, of an antibody selected from antibody 3.1.1 , 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP-675,206), 11.6.1 , 11.7.1, 12.3.1.1 , 12.9.1.1, ipilimumab.

In yet another embodiment, the antibody has heavy and light chain regions having the amino acid sequences of ipilimumab (previously known as MDX-010).

In another embodiment, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 94%, more preferably, at least 95%, even more preferably, at least 99%, sequence identity or sequence similarity over the heavy and light chain full-length sequences, or over the heavy or the light chain, separately, with the sequences of germline Vκ A27, germline Vκ O12, and germline DP50 (which is an allele of the VH 3-33 gene locus). Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy chain sequence of germline DP50 and/or with the light chain sequence of germline A27, or germline O12.

In one embodiment, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 94%, preferably, at least 95%, more preferably, at least 99%, sequence(e.g., amino acid, nucleic acid, or both) identity or sequence similarity over the heavy and light chain variable region sequences, or over the heavy or the light chain variable region sequence, separately, with the sequences of antibody 3.1.1 , 4.1.1 , 4.8.1 , 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1 (CP-675,206), 11.6.1 , 11.7.1 , 12.3.1.1 , 12.9.1.1 , ipilimumab. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy chain and the light chain variable region sequences, or with the heavy chain or the light chain sequence, separately, of an antibody selected from antibody 3.1.1 , 4.1.1, 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP-675,206), 11.6.1 , 11.7.1 , 12.3.1.1 , 12.9.1.1 , ipilimumab. In another embodiment, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 94%, more preferably, at least 95%, even more preferably, at least 99%, sequence identity or sequence similarity over heavy chain variable region sequence with the heavy chain variable sequence of heavy germline DP50 (which is an allele of the VH 3-33 gene locus) or with the light chain variable sequence of germline Vκ A27, or germline Vκ 012. Even more preferably, the antibody heavy chain region sequence shares 100% sequence identity or sequence similarity with the sequence of germline DP50 or with the light chain sequence of germline A27, or germline O12. In one embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, sequence identity or sequence similarity with the heavy chain, the light chain, or both, sequences from FR1 through FR4 with the FR1 through FR4 region sequences of antibody 3.1.1, 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.2.1 (CP- 675,206), 11.6.1 , 11.7.1 , 12.3.1.1 , 12.9.1.1, ipilimumab. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy, light, or both, sequences from FR1 through FR4 with antibody 3.1.1 , 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.6.1 , 11.7.1 , 12.3.1.1, 12.9.1.1 , CP-675,206, and ipilimumab.

In another embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, and most preferably, about 100%, sequence identity or sequence similarity with the heavy chain sequences from FR1 through FR3 with the FR1 through FR3 region sequences of germline DP50.

In yet another embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, and most preferably, about 100%, sequence identity or sequence similarity with the light chain sequences from FR1 through FR4 with the FR1 through FR4 region sequences of germline Vκ A27, or germline Vκ O12.

In one embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, sequence identity or sequence similarity with the heavy chain, the light chain, or both, CDR-1 , CDR-2 and CDR-3 sequences of antibody 3.1.1 , 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1, 11.2.1 (CP-675,206), 11.6.1 , 11.7.1, 12.3.1.1 , 12.9.1.1 , ipilimumab. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy, light, or both, CDR-1 , CDR-2 and CDR-3 sequences with antibody 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, CP-

675,206, and ipilimumab.

In another embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, and most preferably, about 100%, sequence identity or sequence similarity with the heavy chain CDR-1 and CDR-2 sequences with the

CDR-1 and CDR-2 sequences of germline DP50.

In yet another embodiment of the present invention, the antibody shares at least 80%, more preferably, at least 85%, even more preferably, at least 90%, yet more preferably, at least 95%, more preferably, at least 99%, and most preferably, about 100%, sequence identity or sequence similarity with the light chain CDR-1 , CDR-2 and CDR-3 sequences with the CDR-1 , CDR-2 and CDR-3 sequences of germline Vκ A27, or germline Vκ 012.

Examples of antibodies employable in the present invention, and methods of producing them, are described in, among others, U.S. Patent Application No. 09/472,087, now issued as U.S. Patent No. 6,682,736; Int. Appl. No. PCT/USOO/23356 (published March 1 , 2001, as WO

01/14424) (e.g., antibody ipilimumab, also known as MDX-010, Medarex, Princeton, NJ); Int.

Appl. No. PCT/US99/28739 (published June 8, 2000, as WO 00/32231); U.S. Pat. Nos.

5,811 ,097, 5,855,887, 6,051,227, and 6,207,156; each of which is incorporated by reference herein. While information on the amino and nucleic acid sequences relating to these antibodies is provided herein, further information can be found in U.S. Patent No. 6,682,736, as well as

WO 00/37504; the sequences set forth in those applications are hereby incorporated herein by reference.

Certain uses for these antibodies to treat various cancers were discussed in U.S.

Patent Application No. 10/153,382, now published as U.S. Patent Application Publication No. 2003/0086930, which is incorporated by reference as if set forth in its entirety herein.

Characteristics of human anti-CTLA4 antibodies useful in the methods of the invention are extensively discussed in, e.g., U.S. Patent No. 6,682,736, and include antibodies having amino acid sequences of an antibody such as, but not limited to, antibody 3.1.1 , 4.1.1 , 4.8.1 ,

4.10.2, 4.13.1 , 4.14.3, 6.1.1 , 11.6.1 , 11.7.1 , 12.3.1.1 , 12.9.1.1 , CP-675,206, and ipilimumab. The invention also relates to methods using antibodies comprising the amino acid sequences of the CDRs of the heavy and light chains of these antibodies, as well as those comprising changes in the CDR regions, as described in the above-cited applications and patent. The invention also concerns antibodies comprising the variable regions of the heavy and light chains of those antibodies. In another embodiment, the antibody is selected from an antibody comprising the full length, variable region, or CDR, amino acid sequences of the heavy and light chains of antibodies 3.1.1, 4.1.1 , 4.8.1 , 4.10.2, 4.13.1 , 4.14.3, 6.1.1, 11.6.1 , 11.7.1 , 12.3.1.1 , and 12.9.1.1 , CP-675,206, and ipilimumab.

While the anti-CTLA4 antibodies discussed previously herein may be preferred, the skilled artisan, based upon the disclosure provided herein, would appreciate that the invention encompasses a wide variety of anti-CTLA4 antibodies and is not limited to these particular antibodies. More particularly, while human antibodies are preferred, the invention is in no way limited to human antibodies; rather, the invention encompasses useful antibodies regardless of species origin, and includes, among others, chimeric, humanized and/or primatized antibodies. Also, although the antibodies exemplified herein were obtained using a transgenic mammal, e.g., a mouse comprising a human immune repertoire, the skilled artisan, based upon the disclosure provided herein, would understand that the present invention is not limited to an antibody produced by this or by any other particular method. Instead, the invention includes an anti-CTLA4 antibody produced by any method, including, but not limited to, a method known in the art (e.g., screening phage display libraries, and the like) or to be developed in the future for producing an anti-CTLA4 antibody of the invention. Based upon the extensive disclosure provided herein and in, e.g., U.S. Patent No. 6,682,736, to Hanson et al., and U.S. Pat. App. Pub. No. 2002/0088014, one skilled in the art can readily produce and identify an antibody useful for treatment of prostate cancer in combination with a hormonal therapeutic agent using the novel methods disclosed herein. The present invention encompasses human antibodies produced using a transgenic non-human mammal, i.e., XenoMouse™ (Abgenix, Inc., Fremont, CA) as disclosed in the U.S. 6,682,736, to Hanson et al.

Another transgenic mouse system for production of "human" antibodies is referred to as "HuMAb-Mouse™" (Medarex, Princeton, NJ), which contains human immunoglobulin gene miniloci that encodes unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous mu and kappa chain loci (Lonberg et al. Nature 368:856-859 (1994), and U.S. Pat. No. 5,770,429).

However, the invention uses human anti-CTLA4 antibodies produced using any transgenic mammal such as, but not limited to, the Kirin TC Mouse™ (Kirin Beer Kabushiki Kaisha, Tokyo, Japan) as described in, e.g., Tomizuka et al., Proc Natl Acad Sci USA 97:722 (2000); Kuroiwa et al., Nature Biotechnol 18:1086 (2000); U.S. Patent Application Publication No. 2004/0120948, to Mikayama et al.; and the HuMAb-Mouse™ (Medarex, Princeton, NJ) and XenoMouse™ (Abgenix, Inc., Fremont, CA), supra. Thus, the invention encompasses using an anti-CTLA4 antibody produced using any transgenic or other non-human animal. In another embodiment, the antibodies employed in methods of the invention are not fully human, but "humanized". In particular, murine antibodies or antibodies from other species can be "humanized" or "primatized" using techniques well known in the art. See, e.g., Winter and Harris Immunol. Today 14:43-46 (1993), Wright et al. Crit. Reviews in Immunol. 12:125-168 (1992), and US Patent No. 4,816,567, to Cabilly et al, and Mage and Lamoyi in Monoclonal Antibody Production Techniques and Applications pp. 79-97, Marcel Dekker, Inc., New York, NY (1987). Thus, humanized, chimeric antibodies, anti-CTLA4 antibodies derived from any species (including single chain antibodies obtained from camelids as described in, e.g., U.S. Pat. Nos. 5,759,808 and 6,765,087, to Casterman and Hamers), as well as any human antibody, can be combined with a therapeutic agent to practice the novel methods disclosed herein.

As will be appreciated based upon the disclosure provided herein, antibodies for use in the invention can be obtained from a transgenic non-human mammal, and hybridomas derived therefrom, but can also be expressed in cell lines other than hybridomas. Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, NSO, Sp2, HEK, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). Non-mammalian prokaryotic and eukaryotic cells can also be employed, including bacterial, yeast, insect, and plant cells.

Nucleic acid molecules encoding an anti-CTLA4 antibody, and expression vectors comprising these nucleic acid molecules, may be used for transfection of a suitable mammalian, plant, bacterial or yeast host cell. Transformation may be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming plant cells are well known in the art, including, e.g., Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods of transforming bacterial and yeast cells are also well known in the art.

An expression vector may also be delivered to an expression system using DNA biolistics, wherein the plasmid is precipitated onto microscopic particles, preferably gold, and the particles are propelled into a target cell or expression system. DNA biolistics techniques are well-known the art and devices, e.g., a "gene gun", are commercially available for delivery of the microparticles in to a cell (e.g., Helios Gene Gun, Bio-Rad Labs., Hercules, CA) and into the skin (PMED Device, PowderMed Ltd., Oxford, UK).

Various expression systems can be used as well known in the art, such as, but not limited to, those described in, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002). These expression systems include dihydrofolate reductase (DHFR)-based systems, among many others. The glutamine synthetase system of expression is discussed in whole or part in connection with European Patents No. 0216846B1 , No. 0256055B1 , and No. 0323997B1 , and European Patent Application No. EP89303964. In one embodiment, the antibody used is made in NSO cells using a glutamine synthetase system (GS-NSO). In another embodiment, the antibody is made in CHO cells using a DHFR system. Both systems are well-known in the art and are described in, among others, Barnes et al. Biotech & Bioengineering 73:261-270 (2001), and references cited therein. Site directed mutagenesis of the antibody CH2 domain to eliminate glycosylation may be preferred in order to prevent changes in either the immunogenicity, pharmacokinetic, and/or effector functions resulting from non-human glycosylation. Further, the antibody can be deglycosylated by enzymatic (see, e.g., Thotakura et al. Meth. Enzymol. 138:350 (1987)) and/or chemical methods (see, e.g., Hakimuddin et al., Arch. Biochem. Biophys. 259:52 (1987)). Further, the invention encompasses using an anti-CTLA4 antibody comprising an altered glycosylation pattern. The skilled artisan would appreciate, based upon the disclosure provided herein, that an anti-CTLA4 antibody can be modified to comprise additional, fewer, or different glycosylates sites compared with the naturally-occurring antibody. Such modifications are described in, e.g., U.S. Patent Application Publication Nos. 2003/0207336, and 2003/0157108, and International Patent Publication Nos. WO 01/81405 and 00/24893.

Additionally, the invention comprises using an anti-CTLA4 antibody regardless of the glycoform, if any, present on the antibody. Moreover, methods for extensively remodeling the glycoform present on a glycoprotein are well-known in the art and include, e.g., those described in International Patent Publication Nos. WO 03/031464, WO 98/58964, and WO 99/22764, and US Patent Application Publication Nos. 2004/0063911, 2004/0132640, 2004/0142856, 2004/0072290, and US Patent No. 6,602,684 to Umana et al.

Further, the invention encompasses using an anti-CTLA4 antibody with any art-known covalent and non-covalent modification, including, but not limited to, linking the polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in, for example, U.S. Patent Application Publication Nos. 2003/0207346 and 2004/0132640, and U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337.

Additionally, the invention encompasses using an anti-CTLA4 antibody, or antigen- binding portion thereof, chimeric protein comprising, e.g., a human serum albumin polypeptide, or fragment thereof. Whether the chimeric protein is produced using recombinant methods by, e.g., cloning of a chimeric nucleic acid encoding the chimeric protein, or by chemical linkage of the two peptide portions, the skilled artisan would understand once armed with the teachings provided herein that such chimeric proteins are well-known in the art and can confer desirable biological properties such as, but not limited to, increased stability and serum half-life to the antibody of the invention and such molecules are therefore included herein.

Antibodies that are generated for use in the invention need not initially possess a particular desired isotype. Rather, the antibody as generated can possess any isotype and can be isotype switched thereafter using conventional techniques. These include direct recombinant techniques (see, e.g., U.S. Patent 4,816,397), and cell-cell fusion techniques (see e.g., U.S. Patent No. 5,916,771).

The effector function of the antibodies of the invention may be changed by isotype switching to an IgGI , lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM for various therapeutic uses. Furthermore, dependence on complement for cell killing can be avoided through the use of bispecifics, immunotoxins, or radiolabels, for example. Although antibody 4.1.1 , 4.13.1 and 11.2.1 (CP-675,206) are lgG2 antibodies and the sequences of the variable regions of the antibodies are provided herein (Figures 1-3), and in the applications and patents referenced and incorporated herein, it is understood that the full- length sequences of these antibodies are encompassed herein, as well as the use of any antibody comprising the sequences set forth in SEQ ID NOs: 1-36, and further comprising any constant region, regardless of isotype as more fully discussed elsewhere herein. Likewise, any antibody comprising the full-length sequence of ipilimumab, or any portion thereof, including a sequence encoding an antigen-binding portion of ipilimumab, can be used according to the methods of the invention.

Thus, the skilled artisan, once provided with the teachings provided herein, would readily appreciate that the anti-CTLA4 antibody-therapeutic agent combination of the invention can comprise a wide plethora of anti-CTLA4 antibodies.

Further, one skilled in the art, based upon the disclosure provided herein, would understand that the invention is not limited to administration of only a single antibody; rather, the invention encompasses administering at least one anti-CTLA4 antibody, e.g., one of 4.1.1 , 4.13.1, or 11.2.1 (CP-675,206), in combination with a therapeutic agent. Further, any combination of anti-CTLA4 antibodies can be combined with at least one therapeutic agent and the present invention encompasses any such combination and permutation thereof.

IV. Therapeutic Agents The present invention relates to combinations comprising anti-CTLA4 antibody and at least one therapeutic agent, which may be further combined with additional agents and/or therapeutic modalities, e.g., chemotherapy, surgery, radiotherapy, transplantation, and the like, to treat cancer. That is, the patient may be subjected to additional chemotherapy with agents well-known, such as, but not limited to, growth factor inhibitors, biological response modifiers, alkylating agents, intercalating antibiotics, vinca alkaloids, immunomodulators, taxanes, platinum compounds, signal transduction inhibitors, selective estrogen receptor modulators (SERMs), such as, but not limited to, lasofoxifene, and angiogenesis inhibitors.

Co-administration of the antibody with an additional therapeutic agent (combination therapy) encompasses administering a pharmaceutical composition comprising both the anti- CTLA4 antibody and one or more additional therapeutic agents, and administering two or more separate pharmaceutical compositions, one comprising the anti-CTLA4 antibody and the other(s) comprising the additional therapeutic agent(s). Further, although coadministration or combination (conjoint) therapy generally mean that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses simultaneous, sequential or separate dosing of the individual components of the treatment. Additionally, where an antibody is administered intravenously and the anticancer agent is administered orally (e.g., imatinib mesylate, gemcitabine, capecitabine, temozolomide, and the like), it is understood that their combination is preferably administered as two separate pharmaceutical compositions. Therapeutic agents are numerous and have been described in, for instance, U.S.

Patent Application Publication No. 2004/0005318, No. 2003/0086930, No. 2002/0086014, and International Publication No. WO 03/086459, all of which are incorporated by reference herein, among many others. Such therapeutic agents include, but are not limited to, topoisomerase I inhibitors; other antibodies (rituximab, bevacizumab, trastuzumab, anti-IGF 1R antibody [e.g., CP-751,871], anti-CD40 antibody [e.g., CP-870,893], and the like); chemotherapeutic agents such as, but not limited to, imatinib (GLEEVEC), SU11248 (SUTENT; sunitinib), SU12662, SU14813; BAY 43-9006, AG-013736 (axitinib), immunomodulators, including toll-like receptor agonists (e.g., TLR-9 agonist; such as, but not limited to, CPG-7909, also referred to as PF03512676 or PROMUNE), and other immunomodulators, e.g., indoleamine-2,3,-dioxygenase (IDO) inhibitors; selective estrogen receptor modulators (SERMs; e.g., lasofoxifene); taxanes; vinca alkaloids; temozolomide; angiogenesis inhibitors; EGFR inhibitors; VEGF inhibitors; erbB2 receptor inhibitors; antiproliferative agents (e.g., famesyl protein transferase inhibitors, and αvβ3 inhibitors, αvβ5 inhibitors, p53 inhibitors, and the like); immunomodulators; biological response modifiers; cytokines; tumor vaccines; tumor-specific antigens; heat shock protein-based tumor vaccines; dendritic and stem cell therapies; alkylating agents; folate antagonists; pyrimidine antagonists; anthracycline antibiotics; platinum compounds; immune costimulatory molecules (e.g., CD4, CD25, PD-1 , B7-H3, 4-1 BB, OX40, ICOS, CD30, HLA-DR, MHCII, and LFA), and agonist antibodies thereto; among many others.

In one embodiment, the methods of the invention may be further combined with transplantation, e.g., stem cell transplantation, to provide a therapeutic benefit to a patient afflicted with breast cancer. Stem cell transplantation may be performed according to the methods known in the art and may be allogeneic or autologous stem cell transplantation. Additionally, one skilled in the art would appreciate, based upon the disclosure provided herein, that transplantation encompasses adoptive transfer of lymphocytes, either autologous or obtained from an HLA-matched donor. Where the method comprises stem cell transplant, the first dose of the antibody-AI therapy agent combination can be administered after the immune system of the mammal has recovered from transplantation, for example, in the period of from one to 12 months post transplantation. In certain embodiments, the first dose is administered in the period of from one to three, or one to four months post transplantation. Transplantation methods are described many treatises, including Appelbaum in Harrison's Principles of Internal Medicine, Chapter 14, Braunwald et al., Eds., 15th ed., McGraw-Hill Professional (2001), which is hereby incorporated herein by reference.

As pointed out previously herein, there are many chemotherapeutic agents currently available for the treatment of tumors that are suitable for use in the combination therapy of the present invention. For example, alkylating agents are a class of drugs that alkylate DNA, restricting uncoiling and replication of strands. A preferred alkylating agent for use in the methods of the present invention is cyclophosphamide (CYTOXAN). In one embodiment, CP- 675,206 is administered with low-dose cyclophosphamide. Without wishing to be bound by any particular theory, this is because such dose may mediate depletion of Tregs and because CTLA4 blockade appears not to affect Tregs such that these two anti-tumor mechanisms may provide a synergistic therapeutic effect.

Folate antagonists bind to dihydrofolate reductase (DHFR) and interfere with pyrimidine (thymidine) synthesis. Methotrexate and pemetrexed (ALIMTA) are folate antagonists suitable for use in the methods of the present invention. In addition to DHFR, pemetrexed also inhibits thymidylate synthase and glycinamide ribonucleotide formyl transferase, two other folate-dependant enzymes involved in thymidine synthesis. Pyrimidine antagonists inhibit enzymes involved in pyrimidine synthesis. As pyrimidine analogs, they also interfere with DNA production by competing with normal nucleotides for incorporation into the DNA molecule. Pyrimidine antagonists suitable for use in the methods of the present invention include 5-fluorouracil (5-FU); capecitabine (XELODA), a prodrug of 5'-deoxy-5-fluorouridine (5'-FDUR), which is enzymatically converted to 5-FU in vivo; and gemcitabine (GEMZAR).

Anthracycline antibiotics inhibit the uncoiling of DNA by intercalation between DNA strands. Anthracycline antibiotics include doxorubicin hydrochloride (ADRIAMYCIN), epirubicin hydrochloride (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), and idarubicin hydrochloride (IDAMYCIN PFS, ZAVEDOS). Preferred anthracyclines for use with the present invention include doxorubicin and epirubicin.

Platinum compounds exert their anti-neoplastic effect by intercalation and intracalation between DNA strands, which inhibits uncoiling of the DNA. Platinum compounds useful in the methods of the present invention include cisplatin (PLATINOL), oxaliplatin (ELOXATIN), and carboplatin (PARAPLATIN).

Taxanes promote assembly of microtubules while inhibiting their disassembly into tubulin, thereby blocking a cell's ability to break down the mitotic spindle during mitosis. They have demonstrated significant activity against many solid tumors as single agent therapy and in combination with other chemotherapy agents. One embodiment of the combination therapy of the present invention includes the use of one or more taxanes in combination with an IGF- 1 R antibody. Suitable taxanes for use in combination with the IGF-1 R antibody include docetaxel (TAXOTERE) and paclitaxel (TAXOL).

Taxane-derivatives, which may be active in cells resistant to doxorubicin, vinblastine, paclitaxel, docetaxel, and the like, include XRP-9981 (Sanofi Aventis), and are encompassed in the invention.

Vinca alkaloids, like taxanes, are "spindle poisons," acting on the microtubules that form the mitotic spindle. They inhibit mitosis by interfering with microtubule assembly, keeping the spindle from being formed. Vinca alkaloids include vindesine (ELDISINE), vinblastine sulfate (VELBAN), vincristine sulfate (ONCOVIN) and vinorelbine tartrate (NAVELBINE). A preferred vinca alkaloid for use in the methods of the present invention is vinorelbine.

BMS-247550 (ixabepilone) promotes tubulin polymerization and microtubule stabilization, thereby arresting cells in the G2-M phase and inducing tumor cell apoptosis. This agent demonstrates activity against taxane-resistant cells. Analogs of rapamycin, which bind and inhibit the mammalian target of rapamycin (mTOR), are also useful and include, among others, CCI-779 (temsirolimus; Wyeth) and RAD-001 (everolimus, CERTICAN; Novartis).

The camptothecin analogs act through inhibition of topoisomerase I, an enzyme critical for DNA replication and packaging. Levels of topoisomerase I are higher in tumor cells than in normal tissue. A camptothecin analog useful in the methods of the present invention is irinotecan (CAMPTOSAR). Topoisomerase I inhibitors useful in the embodiments of the present invention include 9-aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10-hydroxycamptothecin, lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, and combinations thereof.

Camptothecin derivatives are of particular interest in the combination embodiments of the invention and include camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan and combinations thereof.

A particularly preferred topoisomerase I inhibitor is irinotecan (CAMPTOSAR) Topoisomerase Il inhibitors useful in the combination embodiments of the present invention include aclarubicin, adriamycin, amonafide, amrubicin, annamycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, etoposide, idarubicin, galarubicin, hydroxycarbamide, nemorubicin, novantrone (mitoxantrone), pirarubicin, pixantrone, procarbazine, rebeccamycin, sobuzoxane, tafluposide, valrubicin, and Zinecard (dexrazoxane). Particularly preferred toposimerase Il inhibitors include epirubicin (Ellence), doxorubicin, daunorubicin, idarubicin and etoposide.

Alkylating agents that may be used in the embodiments of the invention include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, AMD-473, altretamine, AP- 5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, chlorambucil, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine, mafosfamide, mechlorethamine, melphalan, mitobronitol, mitolactol, mitomycin C, mitoxatrone, nimustine, ranimustine, temozolomide, thiotepa, and platinum-coordinated alkylating compounds such as cisplatin, carboplatin (PARAPLATIN), eptaplatin, lobaplatin, nedaplatin, oxaliplatin (ELOXATIN, Sanofi), streptozocin, satraplatin and combinations thereof.

Antimetabolites that may be used in combination therapy with CTLA4 antibodies, optionally with one or more other agents include, but are not limited to dihydrofolate reductase inhibitors (such as methotrexate and NeuTrexin (trimetresate glucuronate)), purine antagonists (such as 6-mercaptopurine riboside, mercaptopurine, 6-thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5- fluorouracil (5-FU), pemetrexed disodium (Alimta, LY231514, MTA), capecitabine (Xeloda), cytosine arabinoside, gemcitabine (Gemzar, EIi Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), doxifluridine, carmofur, cytarabine (including ocfosfate, phosphate stearate, sustained release and liposomal forms), enocitabine, 5-azacitidine (Vidaza), decitabine, and ethynylcytidine) and other antimetabolites such as eflornithine, hydroxyurea, leucovorin, nolatrexed (Thymitaq), triapine, trimetrexate.or for example, one of the preferred anti-metabolites disclosed in European Patent Application No. 239362 such as N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid and combinations thereof.

In another embodiment the anti-cancer agent is a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor such as AG-014699, ABT-472, INO-1001 , KU-0687 and GPI 18180.

Microtubule inhibitors that may be used in combination therapy with CTLA4 antibodies, optionally with one or more other agents include, but are not limited to ABI-007, Albendazole, Batabulin, CPH-82, EPO 906 (Novartis), discodermolide (XAA-296), vinfunine and ZD-6126 (AstraZeneca). Antibiotics that may be used in combination therapy with CTLA4 antibodies, optionally with one or more other agent including, but are not limited to, intercalating antibiotics such as actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), peplomycin, and combinations thereof.

Plant derived anti-tumor substances (also known as spindle inhibitors) that may be used in combination therapy with CP-675,206, but are not limited to, mitotic inhibitors, for example vinblastine, vincristine, vindesine, vinorelbine (NAVELBINE), docetaxel

(TAXOTERE), Ortataxel, paclitaxel (including Taxoprexin a DHA/paciltaxel conjugate) and combinations thereof.

Platinum-coordinated compounds include but are not limited to, cisplatin, carboplatin, nedaplatin, oxaliplatin (ELOXATIN), Satraplatin (JM-216), and combinations thereof.

Other agents include alitretinoin, l-asparaginase, AVE-8062 (Aventis), calcitriol (Vitamin D derivative), Canfosfamide (Telcyta, TLK-286), Cotara (1311 chTNT 1/b), DMXAA (Antisoma), exisulind, ibandronic acid, miltefosine, NBI-3001 (IL-4), pegaspargase, RSR13 (efaproxiral), Targretin (bexarotene), tazarotne (Vitamin A derivative), Tesmilifene (DPPE), Theratope, tretinoin, Trizaone (tirapazamine), Xcytrin (motexafin gadolinium) and Xyotax (polyglutamate paclitaxel), and combinations thereof.

In another embodiment of the present invention statins may be used in combination with CP-675,206. Statins (HMG-CoA reductase inhibitors) may be selected from the group consisting of Atorvastatin (Lipitor, Pfizer Inc.), Pravastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Baycol, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin and Niacin (Advicor, Kos Pharmaceuticals), derivatives and combinations thereof.

In a preferred embodiment the statin is selected from the group consisting of atovorstatin and lovastatin, derivatives and combinations thereof. Other agents useful as anti-tumor agents include CADUET, Lipitor and torcetrapib.

In certain embodiments of the invention, the above described methods are combined with a cancer vaccine described, e.g., in Rosenberg, S., Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62 (2000); Logothetis, C. ASCO Educational Book Spring: 300-302 (2000); Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., in, 1997, in Cancer: Principles and Practice of Oncology, pp. 3023-3043, Fifth Edition, DeVita, V. et al., eds. (1997), including a vaccine using autologous or allogeneic tumor cells. Cellular vaccines have been shown to be effective especially when the tumor cells are transduced to express GM-CSF, which is a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90 (80: 3539-43).

Numerous tumor-specific antigens have been identified to date. The study of gene expression and large scale gene expression patterns in various tumors has led to the definition of so called tumor specific antigens (Rosenberg, SA Immunity 10:281-287 (1999)). In many cases, these tumor specific antigens are differentiation antigens expressed in the tumors and in the cell from which the tumor arose, for example melanocyte antigens gp 100, MAGE antigens, Trp-2. The tumor antigen may also include the protein telomerase, which is required for the synthesis of telomeres of chromosomes and which is expressed in more than 85% of human cancers and in only a limited number of somatic tissues (Kim, N et al. Science 266:2011-2013 (1994)). These somatic tissues may be protected from immune attack by various means. More importantly, many of these antigens can be shown to be the targets of tumor specific T cells found in the host, and such tumor-specific antigens can be combined with an anti-CTLA4 antibody to treat cancer by stimulating an anti-tumor response in the host. More particularly, CTLA4 inhibition can render these otherwise non-immunogenic proteins immunogenic thereby mediating an immune response to the tumor cell expressing them. That is, these proteins are normally viewed by the immune system as self antigens and are therefore tolerogenic but such tolerization can be overcome by anti-CTLA4 antibodies.

Tumor-specific antigen can also be "neo-antigens" expressed in cancer cells because of somatic mutations that alter protein sequence or create fusion proteins between two unrelated sequences (i.e. bcr-abl in the Philadelphia chromosome mutation for CML), or idiotype from B cell tumors. Other tumor vaccines can include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Regardless of the source of the tumor-specific antigen, where it is expressed by a tumor cell, co-administration of the antigen with an anti-CTLA4 antibody can render the tumor cell subject to immune attack thus providing a therapeutic benefit. Another form of tumor-specific antigen which can be used in conjunction with CTLA4 antibody administration is a purified heat shock protein (HSP) isolated from the tumor tissue. These heat shock proteins contain fragments of proteins from the tumor cells and are highly efficient at delivery to antigen presenting cells for eliciting tumor immunity (Suot, R & Srivastava, P Science 269:1585-1588 (1995); Tamura, Y. et al. Science 278:117-120 (1997). Thus, administration of an anti-CTLA4 antibody in combination with a tumor HSP can provide an important benefit by mediating an immune response to the tumor. HSP-based tumor vaccines, and methods for producing the same, include, e.g., ONCOPHAGE (HSPPC-96), and AG-858 (HSPPC-70), both from Antigenics (Lexington, MA), among others.

Another therapeutic agent that can be combined with an anti-CTLA4 antibody is a dendritic cell (DC). DCs are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and can be loaded with various protein and peptide antigens, as well as with tumor cell extracts (Nestle, F. et al. Nature Medicine 4:328-332 (1998)). Where the DCs are loaded with tumor cell extracts, the precise nature of the tumor antigen need not be established; rather, the DC cell processes the tumor extract and presents the antigen(s) in the context of MHC. Thus, the nature of the antigen need not be elucidated and the ability of the DCs to present it can be exploited. DCs can also be transduced by genetic means to express desired tumor antigens as well. DCs have also been fused directly to tumor cells for the purposes of immunization (Kugler, A. et al. Nature Medicine 6:332-336 (2000)). As a method of tumor vaccination, DC immunization can be effectively combined with an anti-CTLA4 antibody to mediate a more potent anti-tumor response. See also Oh et al., Cancer Res. 64:2610-2618 (2004) (TARP epitopes and breast cancer); Kontani et al., lnt J Molec Med 12:493-502 (2003) (dendritic ceil vaccine targeting MUC1 mucin); Holmberg & Sandmaier Expert Rev Vaccines 3:269-277 (2004)(vaccination for breast or ovarian cancer). As suggested by data disclosed herein, an immune response to a vaccine-specific antigen may be enhanced by coadministration of anti-CTLA4 antibody. Therefore, the present invention includes a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of an anti-CTLA4 antibody and a therapeutically effective amount of at least one therapeutic agent where the agent comprises a vaccine and/or antigen, or combination of more than one antigen, or a cell presenting an antigen and/or expressing a cytokine, e.g., GM-CSF. That is, useful vaccines may be, without limitation, those comprised of breast cancer- associated antigens (e.g., HER-2/neu, mammaglobin, prostate and breast tumor-associated protein [TARP], MUC1 , CEA, sialyl-Tn and other carbohydrate antigens), other tumor cancer- associated antigens (e.g., p53, telomerase), anti-idiotype antibodies such as 11 D10, as well as vaccines comprising GM-CSF (see, e.g., GVAX, Cell Genesys, Inc., Lapuleucel-T [APC8024] and Sipuleucel-T [APC8015], Dendreon Corp.; and TVAX, Geron Corp.), DNA and cell-based vaccines, dendritic cell vaccines (reviewed in Banchereau & Palucka, Nature Revs. Immunol. 5:296-306 (2005)), recombinant viral (e.g., vaccinia virus) vaccines, and heat shock protein (HSP) vaccines (e.g., vitespen [ONCOPHAGE], Antigenics Inc.). In one embodiment, the present invention encompasses a combination of CP-

675,206 and a tumor antigen. Tumor antigens or tumor-associated antigens (TAAs) include, e.g., cancer-germ cell (CG) antigens (MAGE, NY-ESO-1), mutational antigens (MUM-1 , p53, CDK-4), over-expressed self-antigens (p53, HER2/NEU), viral antigens (from Papilloma Virus, Epstein-Barr Virus), tumor proteins derived from non-primary open reading frame mRNA sequences (NY-ESO1 , LAGE1), Melan A, MART-1 , MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, tyrosinase, gplOO, gp75, HER-2/neι/, c-erb-B2, CEA, PSA, MUC-1, CA-125, Sialyl- Tn (STn), STn-KLH (THERATOPE, Biomira Inc.), TAG-72, KSA (17-1A), PSMA, p53 (point mutated and/or overexpressed), RAS (point mutated), EGF-R, VEGF, GD2, GM2, GD3, Anti- Id, CD20, CD19, CD22, CD36, Aberrant class II, B1 , CD25 30 (IL-2R) (anti-TAC), or HPV. TAAs are discussed, for example, in Palena et al., Adv. Cancer Res. 95:115-145 (2006), and other art-recognized treatises, and include use of gplOO from a non-human species, e.g., mouse gplOO, to increase or enhance an anti-tumor response.

In one embodiment, the TAA comprises at least one characteristic likely to be related to generating or enhancing an anti-tumor immune response. Such characteristics may include, but are not limited to, prevalence, specificity, immunogenicity, and necessity for cell viability or growth. More particularly, prevalence means the antigen is present in most patients with a certain type of cancer. In another embodiment, the more types of cancer cells that express the antigen compared with expression of the antigen by normal cells the more desirable the prevalence would be. In yet another embodiment, the TAA is specific for the cancer cells in that the expression of the TAA is significantly greater on a cancer cell compared with the level of expression of the TAA on an otherwise identical but non-cancer cell, more preferably, the TAA is not detectably expressed on a normal cell but is expressed on most, if not all, cancer cells in a patient. Most preferably, the TAA is unique to a tumor cell and is not expressed in a normal cell. In yet another embodiment, the TAA is immunogenic, e.g., it induces a detectable immune response to a tumor cell. More specifically, the higher the magnitude of the response, the more preferred the TAA. Even more preferably, the TAA induces, increases and/or prolongs a cellular immune response.

In another embodiment, the TAA is vital to cell growth or survival. That is, the TAA is needed by a tumor cell to survive, grow and/or proliferate. Even more preferably, the TAA is vital to a tumor cell such that affecting or inhibiting a biological activity of the TAA prevents or inhibits cell survival or proliferation. Even more preferably, the TAA is vital such that inhibiting or reducing a biological activity of the antigen inhibits development or selection of a tumor cell variant not expressing the antigen or a variant cell expressing a mutant antigen not affected by the anti-tumor immune response to the original TAA. That is, inducing an immune response to the antigen prevents or inhibits cell survival such that a variant antigen that avoids the immune response is not selected.

In one embodiment, CP-675,206 is administered to a patient (e.g., a melanoma patient) previously vaccinated using a tumor vaccine (e.g., a MART-1 antigen), thereby inducing, enhancing and/or prolonging an immune response to the vaccine, thereby providing a therapeutic benefit to the patient. In one aspect, CP-675,206 can be administered with at least one additional therapeutic agent, such as, but not limited to, a chemotherapeutic agent, an immunomodulatory agent (a TLR-9 agonist), an immune enhancing antibody (e.g., an agonist anti-CD40 antibody), among many additional agents.

In another aspect, a vaccines is be administered prior to, or subsequent to, administration of the antibody-therapeutic agent combination, and when chemotherapy is part of the regimen, a vaccine may be administered prior to chemotherapy. In certain embodiments, the antibody-therapeutic agent combination of the invention may also be administered prior to chemotherapy. In yet other embodiments, the treatment can be combined with stem cell transplant. That is, the antibody-chemotherapeutic agent combination can be administered before or after stem cell transplant. A vaccine may also be administered before or after stem cell transplantation and, in certain embodiments, concomitantly with the antibody.

Stem cell transplantation may be performed according to the methods known in the art. Some such methods are described in Appelbaum in Harrison's Principles of Internal Medicine, Chapter 14, Braunwald et al., Eds., 15th ed., McGraw-Hill Professional (2001), which is hereby incorporated herein by reference. Thus, the methods of the present invention relate to the treatment of cancer in a mammal who has undergone stem cell transplantation, which methods comprise administering to the mammal an amount of a human anti-CTLA4 antibody in combination with a therapeutic agent, which combination is effective in treating the cancer in further combination with stem cell transplantation. Where the method comprises stem cell transplant, the first dose of the antibody- therapeutic agent combination can be administered after the immune system of the mammal has recovered from transplantation, for example, in the period of from one to 12 months post transplantation. In certain embodiments, the first dose is administered in the period of from one to three, or one to four months post transplantation. The patient may undergo stem cell transplantation and preparatory treatment(s).

Where an anti-CTLA4 antibody is combined with a standard cancer treatment, such as, inter alia, chemotherapeutic regimes, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr, M. et al. Cancer Research 58: 5301-5304 (1998)). This is because combined use of an anti-CTLA4 antibody and chemotherapy can mediate cell death that is a consequence of the cytotoxic action of most chemotherapeutic compounds. Such cell death likely results in increased levels of tumor-specific antigen in the antigen presentation pathway, and the anti-CTLA4 antibody mediates an increased immune response thereto. Other combination therapies that can result in synergy with anti-CTLA4 enhancement of the immune response through cell death release of tumor antigens are radiation, surgery, and hormone deprivation, among many others. Each of these protocols, and others described elsewhere herein, creates a source of tumor antigen in the host. Likewise, angiogenesis and signal inhibitors can also be combined with an anti-CTLA4 antibody to increase an immune response to a tumor cell since inhibition of angiogenesis or signal transduction can lead to tumor cell death which may feed tumor antigen into host antigen presentation pathways. Therefore, the combination therapies disclosed herein can provide an increased source of tumor-specific antigens thereby providing an increased immune response to the tumor which, in turn, provides a therapeutic benefit to the patient.

The present invention, as noted previously, encompasses a combination of an anti- CTLA4 antibody and at least one signal transduction inhibitor, such as agents that can inhibit EGFR (epidermal growth factor receptor) responses (e.g., EGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors); VEGF (vascular endothelial growth factor) inhibitors (e.g., VEGF receptors and molecules that can inhibit VEGF); and erbB2 receptor (HER2) inhibitors (e.g., small molecules or antibodies that bind to the erbB2 receptor, where exemplary antibodies include trastuzumab and pertuzumab, which is a HER dimerization inhibitor (HDI)).

EGFR inhibitors are described in, for example in International Patent Publication Nos. WO 95/19970, WO 98/14451, WO 98/02434, and U.S. Patent No. 5,747,498, and such substances can be used in the present invention as described herein. EGFR-inhibiting agents include, but are not limited to, the monoclonal antibodies C225, anti-EGFR 22Mab (ImClone Systems Inc., New York, NY), and ABX-EGF (Abgenix Inc., remont, CA), the compounds ZD- 1839 (AstraZeneca), BIBX-1382 (Boehringer Ingelheim), MDX-447 (Medarexjnα, Annandale, NJ), and OLX-103 (Merck & Co., Whitehouse Station, NJ), TP-38 (IVAX), EGFR fusion protein, EGF-vaccine, anti-EGFR immunoliposomes (Hermes Biosciences Inc.), VRCTC-310 (Ventech Research) and EGF fusion toxin (Seragen Inc., Hopkinton, MA). These and other EGFR- inhibiting agents can be used in the present invention in combination with anti-CTLA4 antibody to treat various cancers.

Compounds directed at inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase (TK) represent a relatively new class of antineoplastic drugs that are useful in the method of the present invention. Many human cancers express members of the EGFR family on the cell surface. When a ligand binds to EGFR, it sets off a cascade of cellular reactions that result in increased cell division and influence other aspects of cancer development and progression, including angiogenesis, metastatic spread, and inhibition of apoptosis. EGFR- TK inhibitors may selectively target one of the members of the EGFR family (EGFR (also known as HER1 or ErbB-1), HER2/neu (also known as ErbB-2), HER3 (also known as ErbB- 3), or HER4 (also known as ErbB-4)), or may target two or more of them. EGFR-TK inhibitors suitable for use in the present invention include gefitinib (IRESSA), erlotinib (TARCEVA), Cl- 1033 (Pfizer), GW2016 (GlaxoSmithKline), EKB-569 (Wyeth), PKI-166 (Novartis), CP- 724,714 (Pfizer), CI-1033 (canertinib, Pfizer Inc), and BIBX-1382 (Boeringer-lngelheim). Additional EGFR-TK inhibitors are described in U.S. Patent No. 6,890,924. VEGF inhibitors, for example SU-5416, SU-6668, SU-11248, SlM 2662, SU-14813

(Sugen/Pfizer), as well as AG-013736 (Pfizer) and CP-547,632 (Pfizer, NY) can also be employed in combination with the anti-CTLA4 antibody. VEGF inhibitors are described, for example, in International Patent Application No. PCT/IB99/00797 (filed May 3, 1999), International Patent Publication Nos. WO 95/21613; WO 97/22596 (published June 26, 1997); WO 97/32856 (published Sept. 12, 1997); WO 98/02437 and WO 98/02438 (both published Jan. 22, 1998); WO 98/50356; WO 98/54093 (published Dec. 3, 1998); WO 99/10349 (published March 4, 1999); WO 99/16755 (published April 8, 1999); WO 99/24440; WO 99/61422; U.S. Patent Nos. 5,792,783, 5,834,504; 5,883,113; 5,886,020; 6,177,401 ; 6,235,764; 6,316,429; 6,395,734; 6,492,383; 6,534,524; and 6,653,308. Other examples of specific VEGF inhibitors useful in the present invention are vandetanib (Zactima), sorafenib (Bayer/Onyx), AEE788 (Novartis), AZD-2171 , VEGF Trap (Regeneron/Aventis), vatalinib (also known as PTK-787, ZK-222584; Novartis/Schering AG as described in U.S. Pat. No. 6,258,812), MACUGEN (pegaptanib octasodium, NX-1838, EYE-001 ; Pfizer Inc/Gilead/Eyetech), IM862 (Cytran Inc., Kirkland, WA); neovastat (Aeterna), IMC-1C11 ImClone antibody, bevacizumab (AVASTIN, Genentech, Inc., San Francisco, CA); cetuximab (ERBITUX, ImCIone); and ANGIOZYME, a synthetic ribozyme that cleaves mRNA producing VEGF1 from Ribozyme (Boulder, CO) and Chiron (Emeryville, CA).

ErbB2 receptor inhibitors, such as GW-282974, GW-572016 (lapatinib) (Glaxo Wellcome pic), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc., Woodlands, TX), mapatumumab (HGS-ETR1, Human Genome Sciences, Inc.) an agonist of TRAIL Receptor 1), trastuzumab (HERCEPTIN; Genentech, Inc., San Francisco, CA), pertuzumab (OMNITARG; 2C4; Genentech, a HER2 dimerization inhibitor HDI)); TAK-165 (Takeda), GW-572016 (lapatinib, GlaxoSmithKline), pelitinib (EKB-569 Wyeth), BMS-599626, PKI-166 (Novartis), dHER2 (HER2 Vaccine, Corixa and GlaxoSmithKline), Osidem (IDM-1), APC8024 (HER2 Vaccine, Dendreon), anti-HER2/neu bispecific antibody (Decof Cancer Center), B7.her2.lgG3 (Agensys), AS HER2 (Research Institute for Rad Biology & Medicine), trifunctional bispecific antibodies (University of Munich), mAB AR-209 (Aronex Pharmaceuticals Inc), and 2B-1 (Chiron), can be combined with the anti-CTLA4 antibody to provide effective combination therapy. Other erbB2 receptor inhibitors are described in, for example, International Patent Publication Nos. WO 95/19970 (published July 27, 1995); WO 97/13760 (published April 17, 1997); WO 98/02434 (published January 22, 1998); WO 98/02437 (published January 22, 1998); WO 99/35132 (published July 15, 1999); WO 99/35146 (published July 15, 1999); U.S. Patent Nos. 5,587,458, and 5,877,305. ErbB2 receptor inhibitors useful in the present invention are also described in EP1029853 (published August 23, 2000) and in International Patent Publication No. WO 00/44728, (published August 3, 2000). ErbB2 receptor inhibitors useful in the present invention are also described in United States Patent Nos. 6,465,449, and 6,284,764, and International Application No. WO 2001/98277 each of which are herein incorporated by reference in their entirety. The erbB2 receptor inhibitor compounds and substances described in the aforementioned PCT applications, U.S. patents, and U.S. provisional applications, as well as other compounds and substances that inhibit the erbB2 receptor, can be used with the anti-CTLA4 antibody in combination therapy in accordance with the present invention.

Various other compounds, such as styrene derivatives, have also been shown to possess tyrosine kinase inhibitory properties, and some of tyrosine kinase inhibitors have been identified as erbB2 receptor inhibitors. Other erbB2 Inhibitors are described in European patent publications EP 566,226 A1 (published October 20, 1993), EP 602,851 A1 (published June 22, 1994), EP 635,507 A1 (published January 25, 1995), EP 635,498 A1 (published January 25, 1995), and EP 520,722 A1 (published December 30, 1992). These publications refer to certain bicyclic derivatives, in particular quinazoline derivatives possessing anti-cancer properties that result from their tyrosine kinase inhibitory properties. Also, WO 92/20642 (published November 26, 1992), refers to certain bis-mono and bicyclic aryl and heteroaryl compounds as tyrosine kinase inhibitors that are useful in inhibiting abnormal cell proliferation. WO 96/16960 (published June 6, 1996), WO 96/09294 (published March 6, 1996), WO 97/30034 (published August 21 , 1997), WO 98/02434 (published January 22, 1998), WO 98/02437 (published January 22, 1998), and WO 98/02438 (published January 22, 1998), also refer to substituted bicyclic heteroaromatic derivatives as tyrosine kinase inhibitors that are useful for the same purpose. Other patent applications that refer to anti-cancer compounds are World Patent Application WO 00/44728 (published August 3, 2000), EP 1029853A1 (published August 23, 2000), and WO 01/98277 (published December 12, 2001), all of which are incorporated herein by reference in their entirety. BAY 43-9006 (Onyx Pharmaceuticals) is an inhibitor of both the RAF/MEK/ERK signaling pathway and the VEGFR-2/PDGFR-β signaling cascade. RAF kinase is an enzyme in the RAS pathway and mutations in the RAS gene are associated with approximately 20% of all human cancers, including 90% of pancreatic cancer, 50% of colon cancer and about 30% of non-small cell lung cancer (NSCLC), and BRAF, a specific RAF kinase, has been demonstrated to be mutated in about two-thirds of melanomas and some colorectal cancer (CRC) and other solid tumors. In addition, VEGFR and PDGFR-β play key roles in angiogenesis such that their inhibition decreases angiogenesis. Thus, the present invention encompasses administering a combination comprising an anti-CTLA4 antibody and BAY 43- 9006 to treat cancer. PDGFR inhibitors include but not limited to those disclosed in Int. Pat. Pub. No. WO

01/40217, published June 7, 2001 and Int. Pat. Pub. No. WO 2004/020431 , published March 11 , 2004, the contents of which are incorporated in their entirety for all purposes.

Preferred PDGFR inhibitors include Pfizer's CP-673,451 and CP-868,596 and their pharmaceutically acceptable salts. TIE-2 inhibitors include GlaxoSmithKline's benzimidazoles and pyridines including

GW-697465A such as described in Int. Pat. Pub. Nos. WO 02/044156 published June 6,

2002, WO 03/066601 published August 14, 2003, WO 03/074515 published September 12,

2003, WO 03/022852 published March 20, 2003 and WO 01/37835 published May 31 , 2001. Other TIE-2 inhibitors include Regeneron's biologicals such as those described in Int. Pat. Pub. No. WO 09/611269 published April 18, 1996, Amgen's AMG-386, and Abbott's pyrrolopyrimidines such as A-422885 and BSF-466895 described in Int. Pat. Pub. Nos. WO 09/955335, WO 09/917770, WO 00/075139, WO 00/027822, WO 00/017203 and WO 00/017202.

Use of an angiogenesis inhibitor in combination with an anti-CTLA4 antibody has been discussed previously elsewhere herein. Moreover, an angiogenesis inhibitor includes, but is not limited to, bevacizumab (AVASTIN) which is a humanized antibody to VEGF. Bevacizumab can be used in combination with 5FU, and is indicated as a first-line treatment of patients with metastatic carcinoma of the colon or rectum. Agents that directly target angiogenic factors or their receptors offer the prospect for greater activity in receptor- competent hematologic malignancies by interrupting autocrine receptor signaling. Bevacizumab produces sustained neutralization of circulating VEGF and may be useful for treatment of myelodysplastic syndrome (MDS), lymphoma, acute myeloid leukemia (AML), and solid tumors. Thus, an anti-CTLA4 antibody and bevacizumab combination therapy, in further combination with 5FU and additional chemotherapeutic agents, for treatment of CRC, and other malignancies, is encompassed by the present invention. In another embodiment of the present invention the anti-angiogenesis agent is a protein kinase C Ii such as enzastaurin, midostaurin, perifosine, staurosporine derivative (such as RO 318425, RO317549, RO318830 or RO 318220 (Roche)), teprenone (Selbex) and UCN-01 (Kyowa Hakko).

The receptor tyrosine kinase inhibitors (RTKI) represent a class of synthetic, small molecule inhibitors of angiogenic receptor signaling. The first receptor antagonist to enter clinical testing in hematologic malignancies is SU5416 (Sugen), which impairs ligand-induced autophosphorylation of the VEGFR-1 and VEGFR-2 receptors and c-Kit. SU5416 inhibits VEGF-induced clonogenic response in leukemia cell lines and promotes apoptosis in myeloblasts from AML patients. Other RTKIs, including SU11248 (sunitinib), SU12662, SU14813, (Pfizer Inc.), vatalanib (also known as PTK787/ZK222584; Novartis), and AG- 013736 (Pfizer), can be used in combination with an anti-CTLA4 antibody to treat AML and other receptor-competent hematologic malignancies. CP-675,206 may be combined with sunitinib (SU-11248), among other RTKIs. Thus, the present invention encompasses a combination of an anti-CTLA4 antibody and at least one antiangiogenic and/or signal transduction inhibitor, including, e.g., SU-11248, SU-12662, SU-14813, AG-013736, as well as other angiogenesis and signal transduction inhibitors that are well-known in the art or developed in the future.

As noted previously, the combination therapy methods of the invention can be used with other agents useful in treating abnormal cell growth or cancer, including, but not limited to other agents capable of enhancing antitumor immune responses, such as additional, different, CTLA4 antibodies, and other agents also capable of blocking CTLA4; and antiproliferative agents such as farnesyl protein transferase inhibitors, and αvβ3 inhibitors, such as the αvβ3 antibody VITAXIN, αvβ5 inhibitors, p53 inhibitors, and the like.

Where the antibody of the invention is administered in combination with another immunomodulatory agent, the immunomodulatory agent can be selected for example from the group consisting of a dendritic cell activator such as CD40 ligand and anti-CD40 agonist antibodies, as well as enhancers of antigen presentation, enhancers of T-cell tropism, inhibitors of tumor-related immunosuppressive factors, such as TGF-β (transforming growth factor beta), and IL-10. Preferred anti-CD40 agonist antibodies encompass antibodies disclosed in International Patent Application No. PCT/US02/36107, filed November 8, 2002, now published as International Patent Publication No. WO 03/040170, and U.S. Patent Application No. 10/292,088, filed November 8, 2002, now published as U.S. Patent Publication No. US2003/0211100, including, but not limited to, an antibody having the heavy and light chain amino acid sequence of antibody 3.1.1 , 3.1.1.H-A78T, 3.1.1 H-A78T-V88 A- V97A, 3.1.1 L-L4M-L83V, 3.1.1 H-A78T-V88A-V97A/3.1.1 L-L4M-L83V, 7.1.2, 10.8.3, 15.1.1 , 21.2.1 , 21.4.1 , 22.1.1 , 22.1.1 H-C109A, 23.5.1 , 23.25.1 , 23.28.1 , 23.28.1 H-D16E, 23.29.1 , and 24.2.1.

The present treatment regimens may also be combined with antibodies or other ligands that inhibit tumor growth by binding to IGF-1 R (insulin-like growth factor 1 receptor). Specific anti-IGF-1 R antibodies that can be used in the present invention include those described in International Patent Application No. PCT/US01/51113, filed 12/20/01 , and published as International Patent Publication No. WO02/053596, International Patent Application No. PCT/IB2004/002555, filed August 3, 2004, and published as International Patent Publication No. WO 2005/016967. Preferred anti-IGFR-1 R antibodies encompass an antibody having the heavy and light chain amino acid sequence of, e.g., antibody 2.12.1 , 2.13.2, 2.14.3, 3.1.1 , 4.9.2 and 4.17.3.

Ligands that inhibit signaling via the IGF-1R also encompass small molecules, and other ligands including, inter alia, somavert (PEGVISOMANT), which is a growth hormone analog that inhibits IGF-1 signaling. PEGVISOMANT is conjugated with polyethylene glycol and can be used, among other things, to treat acromegaly. PEGVISOMANT can be co- administered with anti-CTLA4 antibody to treat cancer in that the combination can inhibit tumor growth. Thus, PEGVISOMANT, similarly with anti-IGF-1 R antibodies, can be used in combination with an anti-CTLA4 antibody to treat cancer as disclosed herein.

Additional exemplary therapeutic and/or prophylactic antibodies include, but are not limited to, SYNAGIS (Medlmmune, MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody; REMICADE (infliximab) (Centocor, PA) which is a chimeric anti- TNFα monoclonal antibody; REOPRO (abciximab) (Centocor) which is an anti-glycoprotein llb/llla receptor; ZENAPAX (daclizumab) (Roche Pharmaceuticals, Switzerland) which is a humanized anti-CD25 monoclonal antibody; AVASTIN (bevacizumab); matuzumab (Merck AG), nimotuzumab, panitumumab, EGFR inhibitor antibody (Amgen/Abgenix); ERBITUX (cetuximab). Other examples are a humanized anti-CD18 F(ab')2 (Genentech); CDP860 which is a humanized anti CD18 F(ab')2 (Celltech, UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4 (Progenics/Genzyme Transgenics); OSTAVIR which is a human anti Hepatitis B virus antibody (Protein Design Lab/Novartis); PROTOVIR which is a humanized anti-CMV IgGI antibody (Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murine-anti-TNF-α F(ab')2 (Knoll Pharma/BASF); IC14 which is an anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgGI antibody (Genentech); OVAREX which is a murine anti-CA 125 antibody (Altarex); PANOREX which is a murine anti-17-IA cell surface antigen lgG2a antibody (Glaxo SmithKline/Centocor); BEC2 which is a murine 5 anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/Medlmmune); Campath 1 H/LDP-03 which is a humanized anti CD52 IgGI antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); rituximab (RITUXAN) which is a chimeric anti-CD20 IgGI antibody (Biogen IDEC/Genentech, Roche/Zettyaku); BEXXAR (131-l-tositumomab); belimumab (LymphoStat-B); HuMax-CD20 (HuMax, Genmab); R 1594 (Roche/Genentech); TRU-015 (Trubion Pharmaceuticals); ocrelizumab (PRO 70769); LYMPHOCI DE which is a humanized anti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanized anti- HLA antibody (Protein Design Lab); ONCOLYM (Lym-1) is a radiolabeled murine anti-HLA diagnostic reagent antibody (Techniclone); ABX-IL8 is a human anti-IL8 antibody (Abgenix); anti-CD11a is a humanized IgGI antibody (Genentech/Xoma); ICM3 is a humanized anti- ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDEC Pharm/Mitsubishi); SGN 14, chimeric anti-CD40 antibody (Seattle Genetics); SGN40, humanized anti-CD40 antibody (Seattle Genetics); CHIR-12.12, antagonist anti-CD40 antibody (Chiron); ISF-154 (Ad-CDI 54, Tragen); toralizumab; ABI-793 (Novartis); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti- complement factor 5 (C5) antibody (Alexion Pharm); adalimumab is a human anti-TNF-α antibody (HUMIRA, CAT/Abbott); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α lgG4 antibody (Celltech); LDP-02 is a humanized anti — α4β7 antibody (LeukoSite/Genentech); OrthoClone 0KT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN is a humanized anti VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FoγR) antibody 30 (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 lgG4 antibody (Celltech/Schering); SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies, respectively (SmithKline Beecham); rhuMab-E25 is a humanized anti-lgE IgGI antibody (Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a murine anti CD-147 IgM antibody (Abgenix); BTI- 322 is a rat anti-CD2 IgG antibody (Medimmune/Bio Transplants); Orthoclone/OKT3 is a murine anti-CD3 lgG2a antibody (Ortho Biotech); SIMULECT is a chimeric anti-CD25 IgGI antibody (Novartis Pharm); LDP-01 is a humanized anti-β2-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab')2 (Pasteur- Merieux/lmmunotech); CAT-152 is a human anti-TGF-β2 antibody (Cambridge Ab Tech); and CORSEVIN M is a chimeric anti-Factor VII antibody (Centocor). The above-listed immunoreactive reagents, as well as any other immunoreactive reagents, may be administered according to any regimen known to those of skill in the art, including the regimens recommended by the suppliers of the immunoreactive reagents.

In another embodiment the agent used in conjunction with CP-675,206 is a hepatocyte growth factor receptor (HGFR or c-MET) antagonist.

In another embodiment of the invention, CP-675,206 or ipilimumab is administered with an antibody to MAdCAM to treat cancer. The nucleotide sequence encoding an antibody or other immunoreactive reagent may be obtained from any information available to those of skill in the art (i.e., from Genbank, the literature, or by routine cloning). If a clone containing a nucleic acid encoding a particular antibody or an epitope-binding fragment thereof or other immunoreactive reagent is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof or other immunoreactive reagent is known, a nucleic acid encoding the immunoglobulin or other immunoreactive reagent may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by any method, including, e.g., PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g. a cDNA clone from a cDNA library that encodes the antibody.

The anti-CTLA4 antibody, as well as any other second antibody administered therewith, may also be administered with a cytokine such as, e.g., interleukin (e.g., IL-1α, IL- 1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18), interferon (e.g., IFNα, IFNβ, IFNγ), tumor necrosis factor (e.g., TNFα, TNFβ), G-CSF, GM-CSF, TGF-β, SLC, endothelial monocyte activating protein-2 (EMAP2), MIP-3α, MIP-3β, or an MHC gene, such as HLA-B7.

In one embodiment, CTLA4 blockade is co-administered with IL-15 or an agonist thereof, to increase, induce or prolong an immune response. This is because studies demonstrated that while CTLA4 blockade induced rejection of engrafted islet cells, inhibition of IL-15/IL-15R processes inhibited activation of CD8+ T cells thereby increasing immunogenic tolerance and preventing graft rejection. That is, anti-CTLA4 blockade leads to graft rejection and IL-15-blockade had the opposite effect. See Ferrari-Lacraz et al., Transplantation 82:1510-1517 (2006). Thus, where decreasing immunogenic tolerance is desired, e.g., where inducing an anti-tumor immune response in a patient is desired, coadministration of a CTLA4 antagonist in combination with IL-15, or an agonist of IL-15/IL-15R, may enhance a therapeutic anti-tumor response, and may provide a greater immune response than either agent alone, and more preferably, may provide a synergistic effect greater than the additive effect of each agent combined. Thus, in an embodiment of the invention, combination therapy comprising an anti-CTLA4 antibody and an agonist of the IL-

15/IL-15R signaling pathway (e.g., IL-15, an agonist anti-IL15 antibody, an agonist anti-IL-15R antibody, among others), is provided to induce, increase and/or prolong an immune response.

Additional exemplary cytokines include other members of the TNF family, including, but not limited to, TNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-related activation- induced cytokine (TRANCE), TNF-α-related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT-α, LT-β, OX40L, FasL, CD27L, CD30L, 4-1 BBL, APRIL, LIGHT, TL1 , TNFSF16, TNFSF17, and AITR-L, or a functional portion thereof. See, e.g., Kwon et al., Curr. Opin. Immunol. 11 :340-345 (1999) for a general review of the TNF family.

In one embodiment, interferons and numerous other immune enhancing agents that may be used in combination therapy with CP-675,206 include, but are not limited to interferon alpha, interferon alpha-2a, interferon, alpha-2b, interferon beta, interferon gamma-1a, interferon gamma-1b (Actimmune), or interferon gamma-n1 , PEG lntron A, and combinations thereof. Other agents include interleukin 2 agonists (such as aldesleukin, BAY-50-4798, Ceplene (histamine dihydrochloride), EMD-273063, MVA-HPV-IL2, HVA-Muc-1-IL2, interleukin 2, teceleukin and Virulizin), Ampligen, Canvaxin, CeaVac (CEA), denileukin, filgrastim, Gastrimmune (G17DT), gemtuzumab ozogamicin, Glutoxim (BAM-002), GMK vaccine (Progenies), Hsp 90 inhibitors (such as HspE7 from Stressgen, AG-858, KOS-953, MVJ-1-1 and STA-4783), imiquimod, krestin (polysaccharide K), lentinan, Melacine (Corixa), MelVax (mitumomab), molgramostim, Oncophage (HSPPC-96), OncoVAX (including OncoVAX-CL and OncoVAX-Pr), oregovomab, sargramostim, sizofiran, tasonermin, TheraCys, thymalfasin, pemtumomab (Y-muHMFG1), picibanil, Provenge (Dendreon), ubenimex, WF-10 (Immunokine), Z-100 (Ancer-20 from Zeria), Lenalidomide (REVIMID, Celegene), thalomid (Thalidomide), and combinations thereof.

The present invention includes coadministration of an anti-CTLA4 antibody, or combination of such antibodies, with at least one T cell costimulatory molecule (also referred to herein as a "costim"), including, but not limited to, CD4, CD25, and the like. Costimulatory molecules further include, e.g., PD-1, B7-H3, OX40, ICOS, CD30, HLA-DR, MHCII, and LFA, or agonist antibodies thereto. These and other costims are well-known in the art and have been well characterized as described in, e.g., Schwartz et al., Nature 410:604-608 (2001); Schwartz et al., Nature Immunol. 3:427-434 (2002); and Zhang et al., Immunity 20:337-347 (2004).

In one alternative embodiment of the invention, CP-675,206 or ipilimumab is administered with an antibody to human 4-1BB to treat cancer. Suitable anti-4-1 BB antibodies are described in, e.g., Int. Appl. No. PCT/US2004/033587 (published April 21 , 2005, as WO 2005/035584). One such antibody is BMS66513. In another embodiment of the invention, CP-675,206 or ipilimumab is administered with an antibody to human mucosal addressin cell adhesion molecule (MAdCAM) to treat cancer. Suitable anti-MAdCAM antibodies are described in, e.g., Int. Appl. No. PCT/US2005/000370 (published July 28, 2005, as WO 2005/067620). Preferably, the anti- MAdCAM antibody is an antibody comprising a heavy and light chain variable region amino acid sequence of antibody 1.7.2, 1.8.2, 6.14.2, 6.22.2, 6.34.2, 6.67.1 , 6.73.2, 6.77.1 , 7.16.6, 7.20.5, 7.26.4, and 9.8.2.

In addition to combination of anti-CTLA4 and immunostimulatory compounds to increase an anti-tumor immune response, it would be understood that certain immunosuppressive molecules may inhibit or decrease an anti-tumor response. Accordingly, in another embodiment of the invention, anti-CTLA antibody is administered in combination with an inhibitor of an immunosuppressive molecule. Such immunosuppressive molecules include, but are not limited to, IDO, TGF-β, PD-1, among others. Thus, a combination of anti- CTLA4 blockade and inhibition of another immunoregulatory pathway is encompassed in the present invention, thereby further enhancing the anti-tumor response provided by anti-CTLA4 blockade.

The treatment regimens described herein may be combined with anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix- metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors, can be used in conjunction with the antibody in the method of the invention. Examples of useful COX-II inhibitors include CELEBREX (celecoxib), parecoxib, deracoxib, ABT-963, COX-189 (Lumiracoxib), BMS 347070, RS 57067, NS-398, Bextra (valdecoxib), Vioxx (rofecoxib), SD- 8381 , 4-methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoyl-phenyl)-1 H-pyrrole, 2-(4-ethoxyphenyl)- 4-methyl-1-(4-sulfamoylphenyl)-1 H-pyrrole, T-614, JTE-522, S-2474, SVT-2016, CT-3, SC- 58125 and Arcoxia (etoricoxib). Additonally, COX-II inhibitors are disclosed in U.S. Patent Application Nos. 10/801 ,446 and 10/801,429, the contents of which are incorporated in their entirety for all purposes. In one embodiment the agent is celecoxib as disclosed in U.S. Patent No. 5,466,823, the contents of which are incorporated by reference in its entirety for all purposes. In another embodiment the agent is deracoxib as disclosed in U.S. Patent No. 5,521 ,207, the contents of which are incorporated by reference in its entirety for all purposes. Other useful anti-angiogenic inhibitors used in conjunction with CP-675,206 include aspirin, and non-steroidal anti-inflammatory drugs (NSAIDs) which nonselective^ inhibit the enzymes that make prostaglandins (cyclooxygenase I and II), resulting in lower levels of prostaglandins. Such agents include, but are not limited to, Aposyn (exisulind), Salsalate (Amigesic), Diflunisal (Dolobid), lbuprofen (Motrin), Ketoprofen (Orudis), Nabumetone (Relafen), Piroxicam (Feldene), Naproxen (Aleve, Naprosyn), Diclofenac (Voltaren), lndomethacin (Indocin), Sulindac (Clinoril), Tolmetin (Tolectin), Etodolac (Lodine), Ketorolac (Toradol), Oxaprozin (Daypro) and combinations thereof.

Preferred nonselective cyclooxygenase inhibitors include ibuprofen (Motrin), nuprin, naproxen (Aleve), indomethacin (Indocin), nabumetone (Relafen) and combinations thereof. Other anti-angiogenic compounds include acitretin, angiostatin, aplidine, cilengtide,

COL-3, combretastatin A-4, endostatin, fenretinide, halofuginone, Panzem (2- methoxyestradiol), PF03446962 (ALK-1 inhibitor), rebimastat, removab, Revlimid, squalamine, thalidomide, ukrain, Vitaxin (alpha-v/beta-3 integrin), and zoledronic acid.

Examples of useful matrix metalloproteinase inhibitors are described in International Patent Publication Nos. WO 96/33172; WO 96/27583; WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, European Patent Application Nos. 780386 (published June 25, 1997), 97304971 (filed July 8, 1997), 99308617 (filed October 29, 1999), 606046 (published July 13, 1994), 931788 (published July 28, 1999), 99302232 (filed March 25, 1999), International Application PCT/IB98/01113 (filed July 21 , 1998), Great Britain patent application number 9912961 (filed June 3, 1999), United States Provisional Patent Application No. 60/148,464 (filed August 12, 1999), and U.S. Patent Nos. 5,863,949, and 5,861 ,510.

Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 and/or MMP-9 relative to the other matrix-metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP- 8, MMP-10, MMP-11 , MMP-12, and MMP-13).

Some specific examples of MMP inhibitors useful in the present invention are AG-3340, RO 32-3555, RS 13-0830, ABT-510 (Abbott), ABT 518 (Abbott), Apratastat (Amgen), AZD 8955 (AstraZeneca), Neovostat (AE-941), COL 3 (CollaGenex Pharmaceuticals), doxycycline hyclate, MPC 2130 (Myriad) and PCK 3145 (Procyon), and the compounds recited in the following list: 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]- propionic acid;

3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3- carboxylic acid hydroxyamide; (2R, 3R) 1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl- piperidine-2-carboxylic acid hydroxyamide;

4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide;

3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]- propionic acid;

4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide;

(R) 3-[4-(4-chioro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylic acid hydroxyamide; (2R, 3R) 1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl- piperidine-2-carboxylic acid hydroxyamide;

3-[[4-(4-fiuoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)- amino]-propionic acid;

3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4- yl)-amino]-propionic acid;

3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3- carboxylic acid hydroxyamide;

3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3- carboxylic acid hydroxyamide; and (R) 3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylic acid hydroxyamide; and pharmaceutically acceptable salts and solvates of said compounds.

In another embodiment, the agent is a signal transduction inhibitor. Such inhibitors include small molecules, antibodies, and antisense molecules, and encompass tyrosine kinase inhibitors, serine/threonine kinase inhibitors. More specifically, signal transduction inhibitors include farnesyl protein transferase inhibitors, EGF inhibitors, ErbB-1 (EGFR), ErbB-

2, pan erb, IGF1 R inhibitors, MEK, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, P70S6 kinase inhibitors and inhibitors of the WNT pathway and so called multi- targeted kinase inhibitors In another embodiment, the signal transduction inhibitor is a famesyl protein transferase inhibitor. Famesyl protein transferase inhibitors include the compounds disclosed and claimed in United States Patent 6,194,438, issued February 27, 2002; United States Patent 6,258,824, issued July 10, 2001 ; United States Patent 6,586,447, issued July 1 , 2003; United States Patent 6,071 ,935, issued June 6, 2000; and United States Patent 6,150,377, issued November 21 , 2000. Other famesyl protein transferase inhibitors include AZD-3409 (AstraZeneca), BMS-214662 (Bristol-Myers Squibb), Lonafarnib (Sarasar) and RPR-115135 (Sanofi-Aventis). Each of the foregoing patent applications and provisional patent applications is herein incorporated by reference in their entirety. In another embodiment the signal transduction inhibitor is a GARF inhibitor. Preferred

GARF inhibitors (glycinamide ribonucleotide formyltransferse inhibitors) include Pfizer's AG- 2037 (pelitrexol) and its pharmaceutically acceptable salts. GARF inhibitors useful in the practice of the present invention are disclosed in US Patent No. 5,608,082, which is incorporated in its entirety for all purposes. In another embodiment the signal transduction inhibitor is a MEK inhibitor. MEK inhibitors include Pfizer's MEK1/2 inhibitor PD325901 , Array Biopharm's MEK inhibitor ARRY- 142886, and combinations thereof.

In another embodiment the anti-cancer signal transduction inhibitor is an mTOR inhibitor. mTOR inhibitors include everolimus (RAD001 , Novartis), zotarolimus, temsirolimus (CCI-779, Wyeth), AP 23573 (Ariad), AP23675, Ap23841 , TAFA 93, rapamycin (sirolimus), and combinations thereof.

In another embodiment the anti-cancer signal transduction inhibitor is an Aurora 2 inhibitor such as VX-680 and derivatives thereof (Vertex), R 763 and derivatives thereof (Rigel), and ZM 447439 and AZD 1152 (AstraZeneca), or a Checkpoint kinase 1/2 inhibitors such as XL844 (Exilixis).

In another embodiment the anti-cancer signal transduction inhibitor is an Akt inhibitor (Protein Kinase B) such as API-2, perifosine and RX-0201.

Additionally, other targeted anti-cancer agents include the raf inhibitors sorafenib (BAY- 43-9006, Bayer/Onyx), GV-1002, ISIS-2503, LE-AON and GI-4000, BMS 184476, CCI 779, DTIC, ISIS 2503, ONYX 015, and flavopyridol.

The invention also relates to methods comprising CP-675,206 and cell cycle inhibitors such as the CDK2 inhibitors ABT-751 (Abbott), AZD-5438 (AstraZeneca), alvocidib (flavopiridol, Aventis), BMS-387,032 (SNS 032 Bristol Myers), EM-1421 (Erimos), indisulam (Esai), seliciclib (Cyclacel), BIO 112 (One Bio), UCN-01 (Kyowa Hakko), and AT7519 (Astex Therapeutics) and Pfizer's multitargeted CDK inhibitors PD0332991 and AG24322. The invention also relates to the use of CP-675,206 together with telomerase inhibitors such as transgenic B lymphocyte immunotherapy (Cosmo Bioscience), GRN 163L (Geron), GV1001 (Pharmexa), RO 254020 (and derivatives thereof), and diazaphilonic acid.

The invention also relates to the use of CP-675,206 with hormonal, anti-hormonal, anti-androgenal therapeutic agents such as anti-estrogens including, but not limited to toremifene, raloxifene, anti-androgens such as finasteride, mifepristone, ABARELIX (Praecis),

TRELSTAR, ATAMESTANE (Biomed-777), ATRASENTAN (Xinlay), Bosentan, doxercalciferol, and combinations thereof.

The invention also contemplates the use of CP-675,206 together with gene silencing agents or gene activating agents such as histone deacetylase (HDAC) inhibitors such as suberolanilide hydroxamic acid (SAHA, Merck Inc/Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M-777, MS-275, pivaloyloxymethyl butyrate and PXD-101.

The invention also contemplates the use of CP-675,206 with gene therapeutic agents such as ADVEXIN (ING 201), TNFerade (GeneVec, a compound which express TNFalpha in response to radiotherapy), RB94 (Baylor College of Medicine).

The invention also contemplates CP-675,206 together with ribonucleases such as Onconase (ranpimase).

The invention also contemplates CP-675,206 with antisense oligonucleotides such as bcl-2 antisense inhibitor Genasense (Oblimersen, Genta). The invention also contemplates CP-675,206 together with proteosomics such as PS-

341 (MLN-341) and VELCADE (bortezomib).

The invention also contemplates CP-675,206 together with anti-vascular agents such as Combretastatin A4P (Oxigene).

The invention also contemplates combination of CP-675,206 with traditional cytotoxic agents including DNA binding agents, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, topoisomerase inhibitors and microtubulin inhibitors.

In an embodiment of the invention, an anti-CTLA4 antibody is administered to a patient receiving a chemotherapeutic agent for treatment of cancer. Such chemotherapeutic agents are known in the art and include, but are not limited to: methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrosoureas such as carmustine and lomustine, L-asparaginase, topotecan, nitrogen mustards, Cytoxan, etoposide, BCNU, vinca alkaloids, platinum compounds, mitomycin, gemcitabine, hexamethylmelamine, temsirolimus (CCI-779); lapatinib (GW 572016); RAD-001 (everolimus); XRP-9881 ; ixabepilone (BMS- 247550); pertuzumab (OMNITARG, 2C4); topotecan, tyrosine kinase inhibitors, tyrphostins, imatinib mesylate (GLEEVEC), herbimycin A, genistein, erbstatin, and lavendustin A. In other embodiments, suitable chemotherapeutics include, but are not limited to, alkylating agents: nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU)); alkylsulphonates (e.g., busulfan, treosulfan); triazenes (e.g., dacarbazine); Platinum containing compounds (e.g., cisplatin, carboplatin, aroplatin, oxaliplatin); Plant Alkaloids: Vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine); Taxoids (e.g., paclitaxel, docetaxel; DNA Topoisomerase Inhibitors: epipodophyllins (e.g., etoposide, teniposide, topotecan, 9- aminocamptothecin, camptothecin, crisnatol); mitomycins (e.g., mitomycin C, antimetabolites); anti-folates: DHFR inhibitors (e.g., methotrexate, trimetrexate) IMP dehydrogenase Inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonuclotide reductase Inhibitors (e.g., hydroxyurea, deferoxamine); pyrimidine analogs: uracil analogs (e.g., 5-fluorouracil, floxuridine, doxifluridine, ratitrexed); cytosine analogs (e.g., cytarabine (ara C), cytosine arabinoside, fludarabine); purine analogs (e.g., mercaptopurine, thioguanine); DNA antimetabolites (e.g., 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole, inosine glycodialdehyde, macebecin II, pyrazoloimidazole); Hormonal therapies: Receptor antagonists: Anti-estrogen (e.g., tamoxifen, raloxifene, megestrol); aromatase inhibitors (e.g., exemestane, anastrozole, letrozole); GnRH antagonists (e.g., abarelix, histrelin); selective estrogen receptor modulators (SERMs) (e.g., lasofoxifene); LH-RH agonists (e.g., goserelin, tryptorelin, buserelin, leuprolide acetate); Anti-androgens (e.g., flutamide, bicalutamide, nilutamide, megestrol, cyproterone); Retinoids/Deltoids cis-retinoic acid; vitamin A derivative (e.g., all-trans retinoic acid (ATRA-IV)); vitamin D3 analogs (e.g., EB 1089, CB 1093, KH 1060); Photodvnamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A (2BA-2-DMHA); Cytokines, e.g., IL-1α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-15, IL-18, IFNα, IFNβ, IFNγ, TNFd, TNFβ, G- CSF, GM-CSF, TGF-β, SLC, EMAP2, MIP-3α, MIP-3β, HLA-B7, other members of the TNF family (e.g., TRAIL, TRANCE, TWEAK, CD40L, LT-α, LT-β, OX40L, CD40L, FasL, CD27L, CD30L, 4-1 BBL, APRIL, LIGHT, TL1 , TNFSF16, TNFSF17, and AITR-L, or a functional portion thereof); Angiogenesis Inhibitors: angiostatin (plasminogen fragment), antiangiogenic antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab, BMS-275291, cartilage-derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, CoI 3, combretastatin A-4, endostatin (collagen XVIII fragment), fibronectin fragment, Gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha/beta/gamma, interferon inducible protein (IP- 10), interleukin-12, Kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, MMI 270 (CGS 27023A), MoAb IMC-1C11 , neovastat (Aeterna), NM-3, panzem, PI-88, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prinomastat, prolactin 16kD fragment, proliferin-related protein (PRP), PTK 787/ZK 222594, retinoids, solimastat, squalamine, SS 3304, SU 5416, SU6668, SU11248, SU12662, SU14813, BAY 43-9006, AG-013736, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470, transforming growth factor-beta (TGF-b), vasculostatin, vasostatin (calreticulin fragment), ZD6126, ZD 6474, famesyl transferase inhibitors (FTI), bisphosphonates; Antimitotic agents (e.g., allocolchicine, halichondrin B, colchicine, colchicine derivative, dolstatin 10, maytansine, rhizoxin, thiocolchicine, trityl Cysteine); Other agents: isoprenylation inhibitors; dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion); cell cycle inhibitors (e.g., staurosporine): actinomycins (e.g., actinomycin D, dactinomycin); bleomycins (e.g., bleomycin A2, bleomycin B2, peplomycin); anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone); mTOR inhibitors (e.g., temsirolimus, everolimus); MDR inhibitors (e.g., verapamil); Ca2+ ATPase inhibitors (e.g., thapsigargin); toll-like receptor agonists (e.g., CpG-7909, also known as PF03512676 or PROMUNE; Coley Pharm; reviewed in Krieg, 1998, In: Applied Oligonucleotide Technology, pp. 431-448, CA. Stein and A.M. Krieg, (Eds.), John Wiley and Sons, Inc., New York, NY); costimulatory molecules (e.g., CD4, CD25, PD-1 , B7-H3, 4-1BB, OX40, ICOS, CD30, HLA-DR, MHCII, and LFA, and agonist antibodies thereto); among many other agents known in the art.

Additional anti-cancer agents that may be used in the methods of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amifostine trihydrate; aminoglutethimide; amsacrine; anastrozole; anthramycin; arsenic trioxide; asparaginase; asperlin; azacitidine; azetepa; azotomycin; Bacillus Calmette-Guerin; batimastat; benzodepa; bevacizumab; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; bortezomib; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; capecitabine; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; chlorambucil; cladribine; clodronate; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; darbepoietin; daunorubicin hydrochloride; decitabine; dexormaplatin; dexrazoxane; dezaguanine; dezaguanine mesylate; diaziquone; diethylstilbestrol; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; farmorubicin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; erlotinib; erythropoietin; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; everolimus; exemestane; fadrozole hydrochloride; fazarabine; fenretinide; filgastrim (G-CSF); floxuridine; fludarabine phosphate; fludrocortisone; fluorouracil; fluoxymesterone; flurocitabine; fosquidone; fostriecin sodium; fulvestrant; gefitinib; gemcitabine; gemcitabine hydrochloride; gemtuzumab; goserelin; hydroxyurea; ibritumomab tiuxetan; idarubicin hydrochloride; ifosfamide; ilmofosine; imatinib; interleukin Il (including recombinant interleukin II, or rlL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride; ixabepilone; ketoconazole; lanreotide acetate; lapatinib; letrozole; leucovorin; leuprolide acetate; levamisole; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; medroxyprogesterone; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; mesna; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; octreotide; ormaplatin; oxaliplatin; oxisuran; paclitaxel; pamidronate; pegaspargase; PEG-L-asparaginase; PEG-filgastrim; peliomycin; pentamustine; pentostatin; peplomycin sulfate; perfosfamide; pertuzumab; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfimer; porfiromycin; prednimustine; pemetrexed; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; raltitrexed; riboprine; rituximab; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; somavert (PEGVISOMANT); sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; sunitinib; streptozocin; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; temozolomide; temsirolimus; teniposide; teroxirone; testolactone; thalidomide; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan; toremifene citrate; trastuzumab; tretinoin; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; topotecan; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zolendronate; zorubicin hydrochloride. Other anti-cancer drugs that can be used include, but are not limited to: 20-epi-1 ,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole;

CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors

(ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, A-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;

Ientinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; Ionidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid

A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anti-cancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

In another specific embodiment, a combination comprising an anti-CTLA4 antibody and a second therapeutic agent is administered to a subject receiving radiation therapy for treatment of cancer. For radiation treatment, the radiation can be gamma rays or X-rays. The methods encompass treatment of cancer comprising radiation therapy, such as external- beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. In preferred embodiments, the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In various preferred embodiments, the radiation treatment is administered as internal therapy or brachytherapy wherein a radiaoactive source is placed inside the body close to cancer cells or a tumor mass. Radiation therapy can be administered in accordance to well-known radiotherapy methods for treatment of cancer. The dose and regimen for radiotherapy can be readily determined by one skilled in the art and is based on the stage of the disease, and other factors well-known in the art. For a general overview of radiation therapy, see Hellman, In: Principles of Cancer Management: Radiation Therapy, Chapter 16, DeVita et al., eds., 6th edition, 2001 , J. B. Lippencott Company, Philadelphia.

Co-administration of the antibody with at least one therapeutic agent (combination therapy) encompasses administering a pharmaceutical composition comprising both the anti- CTLA4 antibody and one or more therapeutic agent(s), and administering two or more separate pharmaceutical compositions, one comprising the anti-CTLA4 antibody and the other(s) comprising the therapeutic agent(s). Further, although co-administration or combination (conjoint) therapy generally mean that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses simultaneous, sequential or separate dosing of the individual components of the treatment. Additionally, where an antibody is administered intravenously and the therapeutic agent is administered orally (e.g., exemestane), it is understood that their combination is preferably administered as two separate pharmaceutical compositions.

The present invention also encompasses the administration of other therapeutic agents in addition to the antibody and first therapeutic agent, either concurrently with one or more of those components, or sequentially before and/or after. Such therapeutic agents include cancer vaccines, anti-vascular agents, anti-proliferative agents, antiagionesis agents, signal transduction inhibitors, immunomodulatory agents, cytokines, and palliative agents to provide supportive care, such as, but not limited to, analgesics, anti-emetic agents, anti- diarrheal agents, and steroids. Preferred anti-emetic agents include ondansetron hydrochloride, granisetron hydrochloride, and metoclopramide. Preferred anti-diarrheal agents include diphenoxylate and atropine (LOMOTIL), loperamide (IMMODIUM)1 octreotide (SANDOSTATIN), olsalazine (DIPENTUM), and mesalamine (ASACOL). Preferred steroids include the non-absorbable steroid budesonide (ENTOCORT), and the steroids for systemic administration dexametasone (DECADRON) and prednisone (METICORTEN). Each administration may vary in its duration from a rapid administration to a continuous perfusion. As a result, for the purposes of the present invention, the combinations are not exclusively limited to those that are obtained by physical association of the constituents, but also to those that permit a separate administration, which can be simultaneous or spaced out over a period of time. The compositions according to the invention are preferably compositions which can be administered parentally. However, these compositions may be administered orally or intraperitoneally in the case of localized regional therapies.

As will be appreciated by one of skill in the art, the choice of therapeutic agents to be used in combination with anti-CTLA4 antibodies, and the timing of their use, will be determined in part by the type and stage of the cancer that is being treated.

The methods of the present invention are suitable for use both as first line therapy and second line therapy. Treatment of both early stage and advanced (metastatic) cancers are within the scope of the present invention.

V. Pharmaceutical Compositions

The invention encompasses the preparation and use of pharmaceutical compositions comprising a human anti-CTLA4 antibody of the invention as an active ingredient in combination with a therapeutic agent, e.g., a chemotherapeutic agent, another antibody, an immunostimulatory agent, a signal transduction inhibitor, an angiogenesis inhibitor, preferably, the therapeutic agent is a chemotherapeutic agent. Such a pharmaceutical composition may consist of each active ingredient alone, as a combination of at least one active ingredient (e.g., an effective dose of an anti-CTLA4, an effective does of a therapeutic agent) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional (active and/or inactive) ingredients, or some combination of these. In one embodiment, the antibody is administered parenterally (e.g., intravenously) in an aqueous solution while the therapeutic agent (e.g., a chemotherapeutic agent, a signal transduction inhibitor, an immunomodulator, an angiogenesis inhibitor, and the like) is administered orally in pill/capsule form. However, the skilled artisan would understand, based upon the disclosure provided herein, that the invention is not limited to these, or any other, formulations, doses, routes of administration, and the like. Rather, the invention encompasses any formulation or method of administering an antibody in combination with a therapeutic agent, including, but not limited to, administering each agent separately in a different formulation via a different route of administration, and administering the antibody and the therapeutic agent in a single composition (e.g. where the therapeutic agent is a protein, such as, another antibody, a cytokine, a costim, and the like), in an aqueous composition administered, inter alia, intravenously), among many others. Thus, the following discussion describes various formulations for practicing the methods of the invention comprising administration of any anti-CTLA4 antibody in combination with any therapeutic agent, but the invention is not limited to these formulations, but comprises any formulation as can be readily determined by one skilled in the art once armed with the teachings provided herein for use in the methods of the invention.

In certain embodiments, the antibodies may be present in a neutral form (including zwitter ionic forms) or as a positively or negatively-charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharmaceutically acceptable salt.

The terms "pharmaceutically acceptable salt" refer to a complex comprising one or more antibodies and one or more counterions, where the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

Pharmaceutically acceptable inorganic bases include metallic ions. More preferred metallic ions include, but are not limited to, appropriate alkali metal salts, alkaline earth metal salts and other physiological acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium, manganese, chromium, selenium, tin, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, rubidium, sodium, and zinc, and in their usual valences. Pharmaceutically acceptable acid addition salts of the antibodies of the present invention can be prepared from the following acids, including, without limitation formic, acetic, acetamidobenzoic, adipic, ascorbic, boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic, malonic, edetic, ethylsulfuric, fendizoic, metaphosphoric, succinic, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric, ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic, aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic, lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic, mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic, aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic, ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic, pantothenic, naphthalenesulfonic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuric acid monomethyl ester, cyclohexylaminosulfonic, β-hydroxybutyric, glycine, glycylglycine, glutamic, cacodylate, diaminohexanoic, camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal 5- phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic, galactaric and galacturonic acids.

Pharmaceutically acceptable organic bases include trimethylamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, cyclic amines, quaternary ammonium cations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine, butylamine, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine, glucosamine, histidine, hydrabamine, imidazole, isopropylamine, methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine, triethanolamine, tromethamine, methylamine, taurine, cholate, 6-amino-2-methyl-2-heptanol, 2-amino-2-methyl-1 ,3- propanediol, 2-amino-2-methyl-1-propanol, aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine, phenylcyclohexylamine, 2-(N- morpholino)ethanesulfonic acid, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, Λ/-(2- acetamido)-2-aminoethanesulfonic acid, 1 ,4-piperazinediethanesulfonic acid, 3-morpholino-2- hydroxypropanesulfonic acid, 1 ,3-bis[tris(hydroxymethyl)methylamino]propane, A- morpholinepropanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1 -ethanesulfonic acid, 2-[(2- hydroxy-1 ,1-bis(hydroxymethyl)ethyl)arnino]ethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, 4-(N-morpholino)butanesulfonic acid, 3-(Λ/,Λ/-bis[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, 2-hydroxy-3-

[tris(hydroxymethyl)methylamino]-1 -propanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1 -(2- hydroxypropanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, Λ/,Λ/-bis(2-hydroxyethyl)glycine, N-(2- hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N-[tris(hydroxymethyl)methyl]-3- aminopropanesulfonic acid, N-tris(Hydroxymethyl)methyl-4-anninobutanesulfonic acid, N-(1 ,1- dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 2-

(cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesuifonic acid, 3-(cyclohexylamino)-1-propanesulfonic acid, Λ/-(2-acetamido)iminodiacetic acid, 4- (cyclohexylamino)-i-butanesulfonic acid, Λ/-[tris(hydroxymethyl)methyl]glycine, 2-amino-2- (hydroxymethyl)-1,3-propanediol, and trometamol.

The anti-CTLA4 antibody used in the methods of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion. The antibodies and therapeutic agents used in the present invention may be in a variety of forms. These include, for example, liquid, semi solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the therapeutic agent, the intended mode of administration and the therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The antibodies can be administered by a variety of methods known in the art, including, without limitation, oral, parenteral, mucosal, by-inhalation, topical, buccal, nasal, and rectal. For many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, intravenous or infusion. Non-needle injection may be employed, if desired. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

In certain embodiments, the antibody may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York (1978).

Transdermal delivery of biological molecules, including antibodies, cytokines, and the like, includes microneedles to create micron-sized temporary transport pathways in the skin through which large molecules can be transported. See, e.g., Banga, In: Therapeutic peptides and proteins: Formulation, processing and delivery systems, 2nd ed., Taylor & Francis (Eds.), 2006; Martanto et al., J. Controlled Release 112:357-361 (2006). In one embodiment, microneedles include those composed of silicon, metal or a polymer. In another embodiment, maltose microneedles which dissolve in the skin may be used to administered the compositions of the invention, including anti-CTLA4 antibody, anti-CD40, anti-IGF-1R antibody, rituximab, trastuzumab, IL-15, and the like.

In yet another embodiment, nucleic acid, including nucleic acids encoding a protein, preferably, anti-CTLA4 antibody (e.g., CP-675,206, ipilimumab, and the like), or an ODN, can be delivered transdermal^ using biolistics to provide expression of the antibody or delivery of the ODN in a micromilieu where antigen presenting cells (e.g., dendritic cells) and/or T regulatory cells are present (e.g., Tregs in lymph nodes). Gold microparticles comprising nucleic acids encoding anti-CTLA4 antibody and/or comprising an ODN or nucleic acids encoding a protein of interest, are propelled through the skin using, for example, a "gene gun" (PMED Device, e.g., model ND-10, PowderMed Ltd., Oxford, UK). See, e.g., US Patent Nos. 6,194,389; 6,168,587; 5,478,744. In one embodiment, a synthetic oligodeoxynucleotide (ODN) toll-like receptor 9 agonist (e.g., PF03512676) may be administered using biolistics such that a microparticle comprising the nucleic acid encoding the antibody and a microparticle comprising the ODN, or a microparticle comprising both, are both delivered transdermally. In another embodiment, a nucleic acid encoding a TAA (e.g., gplOO, tyrosinase, MAGE, MART-1 , NY- ESO-1, and the like) is delivered transdermally using biolistics such that the TAA is expressed in and presented by APCs transfected with the nucleic acid. In yet another embodiment, various combinations of an antigen (e.g., TAA), an antibody (anti-CTLA4, anti-CD40, anti-CD20, anti-HER2, etc.), a cytokine (e.g., GM-CSF, IL-15, TNFα, among others), an ODN (e.g., PF03512676), a costimulatory molecule (e.g., PD-1, OX40, 4-1BB, etc.), and the like, may be delivered using biolistics to induce, enhance or prolong and anti-tumor response in a patient.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

In one embodiment, the antibody is administered in a formulation as a sterile aqueous solution having a pH that ranges from about 5.0 to about 6.5 and comprising from about 1 mg/ml to about 200 mg/ml of antibody, from about 1 millimolar to about 100 millimolar of histidine buffer, from about 0.01 mg/ml to about 10 mg/ml of polysorbate 80, from about 100 millimolar to about 400 millimolar of trehalose, and from about 0.01 millimolar to about 1.0 millimolar of disodium EDTA dihydrate. Exemplary formulations encompass, but are not limited to, such formulations described in PCT/US2006/007551 filed March 2, 2006, now published as WO 2006/096488 on Sept. 14, 2006, and PCT/US2006/007555 filed March 2, 2006, now published as WO 2006/096491 on Sept. 14, 2006.

In another embodiment, the antibody is administered in an intravenous formulation as a sterile aqueous solution containing 5 mg/m, or more preferably, about 10 mg/ml, or yet more preferably, about 15 mg/ml, or even more preferably, about 20 mg/ml of antibody, with sodium acetate, polysorbate 80, and sodium chloride at a pH ranging from about 5 to 6. Preferably, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg/ml of antibody, with 20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium chloride at pH 5.5. Further, a solution comprising an anti-CTLA4 antibody can comprise, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and any combination thereof, and many other compounds known in the relevant art.

The compositions of the present invention optionally may further comprise a pharmaceutically acceptable antioxidant in addition to a chelating agent. Suitable antioxidants include, but are not limited to, methionine, sodium thiosulfate, catalase, and platinum. For example, the composition may contain methionine in a concentration that ranges from 1 mM to about 100 mM, and in particular, is about 27 mM.

In one embodiment, part of the dose is administered by an intraveneous bolus and the rest by infusion of the antibody formulation. For example, a 0.01 mg/kg intravenous injection of the antibody may be given as a bolus, and the rest of a predetermined antibody dose may be administered by intravenous injection. A predetermined dose of the antibody may be administered, for example, over a period of an hour and a half to two hours to five hours.

With regard to a therapeutic agent, where the agent is, e.g., a small molecule, it can be present in a pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active therapeutic agents. Particularly contemplated additional agents include anti-emetics, anti-diarrheals, chemotherapeutic agents, cytokines, and the like.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture.

Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n- propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 2O0C) and which is liquid at the rectal temperature of the subject (i.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or gel or cream or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e. such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations as discussed below. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

A composition of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. The active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, (1978). Pharmaceutical compositions are preferably manufactured under GMP conditions.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1 ,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form. Low boiling propellents generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non- ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985), which is incorporated herein by reference.

In one embodiment of the invention, the composition comprising CP-675,206 comprises a sterile solution comprising 20 mM histidine buffer, pH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA hydrate. In one aspect, CP-675,206 is packaged in clear glass vials with a rubber stopper and an aluminum seal. In another aspect, the vial contains about 20 mg/ml of CP-675,206 with a nominal fill of about 400 mg per vial. The anti-CTLA4/therapeutic agent active ingredient combination of the invention, can be administered to an animal, preferably a human. The precise dosage of each active ingredient administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. The anti-CTLA4 antibody may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. The therapeutic agent, preferably, a chemotherapeutic agent, may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

The antibody and therapeutic agent can be co-administered in that they can be administered separately, on different dates or at different times of the day, as well as simultaneously or on the same date. Co-administration thus encompasses any temporal combination of administration of the antibody and the therapeutic agent such that administration of the two agents mediates a therapeutic benefit to the patient that is detectably greater than administration of either agent in the absence of the other.

An antibody-therapeutic agent combination of the invention may be co-administered with numerous other compounds (other antihormonal therapy agents, cytokines, chemotherapeutic and/or antiviral drugs, among many others). Alternatively, the compound(s) may be administered an hour, a day, a week, a month, or even more, in advance of the antibody-therapeutic agent combination, or any permutation thereof. Further, the compound(s) may be administered an hour, a day, a week, or even more preferably, after administration of radiation, stem cell transplant, or administration of any therapeutic agent (e.g., cytokine, chemotherapeutic compound, and the like), or any permutation thereof. The frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds, and the like.

Vl. Kits

The invention includes various kits which comprise a therapeutically effective amount of a human anti-CTLA4 antibody of the invention and a therapeutically effective amount of a therapeutic agent, along with an applicator and instructional materials which describe use of the combination to perform the methods of the invention. Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the invention.

The invention includes a kit for treatment of breast cancer in a patient in need thereof. The kit includes a human anti-CTLA4 antibody of the invention and at least one therapeutic agent. The inhibitor encompasses, but is not limited to, a chemotherapeutic, an antibody, a cytokine, a vaccine, an immunomodulator, among many others. The kit further comprises an applicator, including, but not limited to, a syringe, for administration of the components of the kit to a patient. Further, the kit comprises an instructional material setting forth the pertinent information for the use of the kit to treat breast cancer in the patient. More preferably, the kit comprises at least one anti-CTLA4 antibody selected from an antibody having the heavy and light chain amino acid sequence of antibody 4.1.1 , 4.8.1 , 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1 (CP-675,206), 11.6.1 , 11.7.1., 12.3.1.1, 12.9.1.1 , and ipilimumab, even more preferably, the antibody is an antibody having the heavy and light chain amino acid sequence of antibody 4.13.1 , 11.2.1 (CP-675,206), and ipilimumab (MDX- 010). Preferably, the antibody is an antibody having the heavy and light sequence of 11.2.1 (CP-675,206). Even more preferably, the anti-CTLA4 antibody is CP-675,206 or ipilimumab. Most preferably, the antibody is CP-675,206.

The kit can comprise any number of additional therapeutic agents for treatment of cancer. Such agents are set forth previously and include chemotherapeutic compounds, cancer vaccines, signal transduction inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines, among many others.

The invention also relates to an article of manufacture (e.g., dosage form adapted for i.v. administration) comprising a human anti-CTLA4 antibody in the amount effective to treat cancer (e.g., more than 10 mg/kg, at least 15 mg/kg, or 15 mg/kg) and a therapeutically effective amount of a second therapeutic agent. In certain embodiments, the article of manufacture comprises a container or containers comprising a human anti-CTLA4 antibody, the therapeutic agent, and a label and/or instructions for use to treat cancer.

In one embodiment, the therapeutic agent is at least one agent selected from a hormonal therapy agent, a taxane, a heat shock protein, a topoisomerase inhibitor, a hormonal therapy agent (e.g., leuprolide), a chemotherapeutic compounds, a vinca alkaloid, a platinum compound, a cancer vaccine, a tumor-specific antigen, an angiogenesis inhibitor, a signal transduction inhibitor, agents useful in treating abnormal cell growth or cancer, antibodies or other ligands that inhibit tumor growth by binding to IGF-1 R, and cytokines, among others. The kit can comprise any number of additional therapeutic agents for treatment of cancer. Such agents are set forth previously and include chemotherapeutic compounds, cancer vaccines, signal transduction inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies or other ligands that inhibit tumor growth by binding to IGF-1 R, and cytokines, and palliative care agents (e.g., anti-diarrheals, anti-emetics, etc.) among many others.

In another embodiment of the invention, the kit is for treatment of breast cancer. The kit comprises an anti-CTLA4 antibody and a taxane. In one aspect, the taxane is docetaxel or paclitaxel. In another aspect, the treatment is first line therapy for the treatment of patients with locally advanced or metastatic triple receptor negative breast cancer.

In one embodiment, the kit is for treatment of ovarian cancer. In one aspect, the kit comprises an anti-CTLA4 antibody and a therapeutic agent, where the therapeutic agent is paclitaxel. In another aspect, the kit further comprises carboplatin. In yet another aspect, the kit is for first-line treatment of advanced carcinoma of the ovary. In another aspect, the kit is for second line treatment of a patient who has progressed following prior paclitaxel-based therapy.

In an embodiment of the invention, the kit is for treatment of non-small cell lung cancer. In one embodiment, the kit comprises an anti-CTLA4 antibody and a chemotherapeutic agent, wherein the therapeutic agent is selected from the group consisting of a platin (e.g., carboplatin (PARAPLATIN), paclitaxel (TAXOL)), docetaxel (TAXOTERE), sunitinib (SU11248), erlotinib (TARCEVA), bevacizumab (AVASTIN), pemetrexed (ALIMTA), and PF03512676 (CpG-7909).

In one embodiment, kit is for first line therapy of locally advanced Stage IMb or metastatic Stage IV NSCLC where the kit comprises an anti-CTLA4 antibody and a platin. In another embodiment, the kit comprises carboplatin and paclitaxel. In another embodiment, the kit comprises the antibody and bevacizumab. In a further embodiment, the kit comprises the antibody and PF03512676. In another embodiment of the invention, the kit comprises the antibody and sunitinib.

In one embodiment, the kit is for second line treatment of Stage IHb or metastatic Stage IV NSCLC after failure of platinum-based therapy. In one embodiment, the kit comprises an anti-CTLA4 antibody and docetaxel. In another embodiment, the kit comprises the antibody and erlotinib. In yet another embodiment, the kit comprises the antibody and pemetrexed. In yet another embodiment, the kit comprises an anti-CTLA4 antibody.

In another embodiment of the invention, the kit is for third line treatment of locally advanced Stage IHb or metastatic Stage IV NSCLC. The kit comprises an effective amount of an anti-CTJ_A4 antibody. In one aspect, the kit is for treatment of cancer after failure of prior platinum-based chemotherapy and EGFR inhibition-based therapy.

In one embodiment, the kit is for treatment of pancreatic cancer. The kit comprises an anti-CTLA4 antibody and a therapeutic agent, wherein the agent is gemcitabine. In one aspect, the treatment is first line therapy for locally advanced, non-resectable Stage Il or

Stage III, or metastatic Stage IV adenocarcinoma of the pancreas.

In one embodiment, the kit is for treatment of melanoma and comprises an effective amount of an anti-CTLA4 antibody and an effective amount of IFNα. In one aspect, the treatment is adjuvant therapy for Stage ll/lll melanoma. In an embodiment of the present invention, the kit is for treatment of colorectal carcinoma. In one aspect, the kit comprises an effective amount of an anti-CTLA4 antibody and an effective amount of each of fluorouracil, leucovorin and oxaliplatin (FOLFOX). In another aspect, the kit comprises an effective amount of an anti-CTLA4 antibody, fluorouracil, leucovorin and irinotecan (FOLFIRI). In one aspect, the treatment is first line therapy for metastatic CRC. In another aspect, the therapy is adjuvant therapy for Stage III colon cancer in a patient who has undergone resection of a primary tumor.

In another embodiment, the kit is for treatment of CRC and comprises an effective amount of an anti-CTLA4 antibody and an effective amount of capecitabine (XELODA). In one aspect, the treatment is first line therapy for a patient intolerant of therapy comprising oxaliplatin (ELOXATIN) or irinotecan (CAMPTOSAR).

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLES

EXAMPLE 1 :

CP-675.206 in Combination with rituximab for First-line Treatment of Indolent Non-Hodqkin's Lymphoma

Patients having indolent non-Hodgkin's lymphoma are treated with CP-675,206 and rituximab. Indolent Non-Hodgkin's Lymphomas (NHLs) are slow growing forms of lymphoma and encompass what were previously referred to as low grade and some categories of intermediate grade NHL in the Working Formulation. Non-Hodgkin's lymphoma (NHL) with at least one lesion that can be accurately measured in two dimensions and whose size is ≥ 2 cm x 1 cm by conventional CT scan or ≥ 1 cm x 1 cm by spiral CT scan are given standard rituximab (RITUXAN) antibody therapy per established protocols.

The patient is further administered a single IV infusion (100 mL/hr) of CP-675,206 at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of CP-675,206 dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Rituximab is administered via intravenous infusion at about 375 mg/m2 once weekly for about four or eight doses. More preferably, rituximab is administered on day 1 of each cycle of chemotherapy.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of either antibody. Premedication is recommended but not required.

Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

Rituximab and anti-CTLA4 antibody are administered either sequentially or simultaneously either once, or repeatedly, as determined.

Following combination treatment with rituximab and anti-CTLA4 antibody, anti-CTLA4 antibody is administered as single agent (SA) therapy as described supra for combination therapy.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate. For all patients, Eastern Cooperative Oncology Group performance status (ECOG

PS), vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained.

Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose. The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 2:

CP-675.206 in Combination with rituximab for Second-line Treatment of Aggressive Non-Hodqkin's Lymphoma

Patients having aggressive, CHOP-R refractory NHL with at least one lesion that can be accurately measured in two dimensions and whose size is ≥ 2 cm x 1 cm by conventional CT scan or ≥ 1 cm x 1 cm by spiral CT scan are given standard rituximab antibody therapy per established protocols. The patient is further administered a single IV infusion (100 mL/hr) of CP-675,206 as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti- CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter. The patient is administered rituximab according to an art-recognized dosing regimen.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required. Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days. Rituximab (e.g., RITUXAN) and the anti-CTLA4 antibody are administered either sequentially or simultaneously either once, or repeatedly, as determined.

Following combination treatment with RITUXAN and the anti-CTLA4 antibody, the CTLA4 antibody is administered as described supra.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained.

Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 3:

CP-675.206 in Combination with 5-fluorouracil or capecitabine for Adjuvant or First-line Treatment of Colorectal Carcinoma

Patients having colorectal carcinoma (CRC) and intolerant of oxaliplatin (ELOXATIN) or irinotecan (CAMPTOSAR) with at least one lesion that can be accurately measured in two dimensions and whose size is ≥ 2 cm x 1 cm by conventional CT scan or ≥ 1 cm x 1 cm by spiral CT scan are given standard chemotherapy using 5FU (fluorouracil) or XELODA

(capecitabine) per established protocols.

The patient is further administered a single IV infusion (100 mL/hr) of CP-675,206 as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-

CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression.

Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

Capecitabine for second line therapy or 5-fluorouracil for adjuvant therapy is administered sequentially or simultaneously with CP-675,206 either once, or repeatedly, as determined. Capecitabine and fluorouracil are administered according to well-known protocols, for examples, those disclosed in the FDA-approved label for each compound.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained.

Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 4: CP-675,206 in Combination with FOLFOX for Adjuvant or First Line Treatment of

Colorectal Carcinoma

Following surgery/radiotherapy patients having colorectal carcinoma (CRC) with at least one lesion that can be accurately measured in two dimensions and whose size is ≥ 2 cm x 1 cm by conventional CT scan or ≥ 1 cm x 1 cm by spiral CT scan are given standard chemotherapy using FOLFOX (fluorouracil, leucovorin and oxaliplatin) per established protocols.

The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every

28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

FOLFOX or FOLFIRI therapy is administered according to standard protocols. Briefly, for FOLFOX4, each chemotherapy cycle comprises a two hour infusion of 85 mg/m2 oxaliplatin (ELOXATIN, Sanofi) on day 1 followed by a two hour infusion of 200 mg/m2 leucovorin on days 1 and 2, followed by a bolus of 400 mg/m2 of fluorouracil on days 1 and 2 and then a twenty-two hour infusion of 600 mg/m2 of fluorouracil administered over two consecutive days (referred to as the De Gramont schedule). Chemotherapy is repeated every two weeks. In an exemplary regimen, FOLFIRI is administered as a ninety minute infusion of 180 mg/m2 irinotecan on day 1, a two hour infusion of folinic acid (leucovorin) at 400 mg/m2 on day 1 , and 400-500 mg/m2 fluorouracil IV bolus after leucovorin on day 1, followed by fluorouracil at 2400-3000 mg/m2 by continuous i.v. over forty-six hours starting on day one. The chemotherapy cycle is repeated every two weeks. Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

FOLFOX therapy is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 5:

CP-675.206 and Gemcitabine for Treatment of Pancreatic Cancer Following surgery/radiotherapy, if any, patients having pancreatic cancer with at least one lesion that can be accurately measured in two dimensions and whose size is ≥ 2 cm x 1 cm by conventional CT scan or ≥ 1 cm x 1 cm by spiral CT scan are given standard chemotherapy comprising gemcitabine (e.g., GEMZAR) per established protocols.

The patient is further administered a single IV infusion of anti-CTLA4 antibodies as described herein at a dose of at least about 10 mg/kg, preferably, at about 15 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-CTLA4 antibody dose, if desired, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Gemcitabine is administered according to standard protocols. More particularly, gemcitabine (GEMZAR, Lilly) is administered by intravenous infusion at a dose of 1000 mg/m2 over 30 minutes once weekly for up to 7 weeks (or until toxicity necessitates reducing or holding a dose) followed by a week of rest from treatment. Subsequent cycles should consist of infusions once weekly for 3 consecutive weeks out of every 4 weeks . CP-675,206 is administered after the initial gemcitabine cycle (seven weeks on and one week rest) and once every three weeks thereafter.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Where appropriate, antibody dose is escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate. For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained.

Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 6: CP-675.206 in Combination with imatinib mesylate for First-line Treatment of

Chronic Myeloid Leukemia

Patients having chronic myeloid leukemia (CML) are given standard chemotherapy using imatinib mesylate (GLEEVEC) per established protocols.

The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every

28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Imatinib mesylate (GLEEVEC, Novartis) is administered daily at about 400 mg/day for patients in chronic phase CML and 600 mg/day for adult patients in accelerated phase or blast crisis. Imatinib treatment may be continued as long as there is no evidence of progressive disease or unacceptable toxicity. Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days. lmatinib mesylate is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method. EXAMPLE 7:

CP-675,206 in Combination with Imatinib Mesylate (GLEEVEC) for First-line Treatment of Chronic Lymphocytic Leukemia

Patients having chronic lymphocytic leukemia (CLL) are given standard chemotherapy using imatinib mesylate (GLEEVEC) per established protocols. The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter. Imatinib mesylate (GLEEVEC, Novartis) is administered daily at about 400 mg/day or 600 mg/day, and may be continued as long as there is no evidence of progressive disease or unacceptable toxicity.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required. Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

GLEEVEC is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTTj, prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method. EXAMPLE 8:

CP-675.206 in Combination with carboplatin and paclitaxel for First-line Treatment of Advanced Ovarian Carcinoma

Patients having advanced carcinoma of the ovary are given standard chemotherapy using carboplatin and paclitaxel per established protocols. The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 3 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days with escalation of the anti-CTLA4 antibody dose, that is, 6 mg/kg, 10 mg/kg and 15 mg/kg, every 28 days thereafter for maximum of 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter. In an exemplary regimen, paclitaxel is administered every three weeks at 175 mg/m2 by i.v. infusion over ninety minutes followed by carboplatin (area under the time-concentration curve [AUC] of 5) administered i.v. over thirty to sixty minutes.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Doses are escalated using an accelerated titration design utilizing a dose-doubling schema with 3-6 subjects per cohort. Within each new cohort there is no required waiting period between subjects. Subsequent cohorts may not be opened until the first subject at the current dose level has been observed for 21 days and subsequent subjects have been observed for 14 days.

Carboplatin and paclitaxel combination is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method. EXAMPLE 9:

CP-675,206 in Combination with carboplatin and paclitaxel and either bevacizumab. PF03512676, or sunitinib, for First-line Treatment of Non-Small Cell Lung Cancer

Patients having locally advanced Stage IHb or mestatatic Stage IV NSCLC are given standard chemotherapy using carboplatin and paclitaxel per established protocols. The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 15 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days for approximately 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

The patient is further administered either bevacizumab (AVASTIN), PF03512676, or sunitinib per established protocols. Bevacizumab may be administered at about 5 mg/kg every fourteen days by i.v. infusion until disease progression is detected. Sunitinib may be administered as one 50 mg oral dose taken daily, on a schedule of four weeks on treatment followed by two weeks off. Sunitinib may be taken with or without food. Dose increase or reduction of 12.5 mg increments is recommended based on individual safety and tolerability. An exemplary carboplatin and paclitaxel dosing regimen is as provided supra.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Carboplatin and paclitaxel combination is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined. Similarly, bevacizumab, sunitinib, or PF03512676 is administered sequentially or simultaneously with anti-CTLA4- carboplatin-paclitaxel, either once, or repeatedly, as indicated. CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein,

TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained.

Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method. EXAMPLE 10:

CP-675,206 in Combination with either docetaxel, erlotinib or pemetrexed, for Second Line Treatment of Non-Small Cell Lung Cancer

Patients having locally advanced Stage IHb or mestatatic Stage IV NSCLC, and which have previously failed platinum-based chemotherapy, are given standard chemotherapy using one of either docetaxel (TAXOTERE), erlotinib (TARCEVA), or pemetrexed (ALIMTA) per established protocols.

The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 15 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days for approximately 12 cycles in the absence of intolerable toxicity or disease progression. Preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Docetaxel (TAXOTERE, Sanofi-Aventis) is administered at about 75 mg/m2 by i.v. infusion over one hour every three weeks. Erlotinib (TARCEVA, OSI Pharms.) is administered as a daily dose of about 150 mg taken at least one hour or two hours after ingestion of food. Treatment should continue until disease progression or unacceptable toxicity occurs. Pemetrexed (ALIMTA, Lilly) is administered at a dose of about 500 mg/m2 administered as an i.v. infusion over 10 minutes on day 1 of each 21 -day cycle. Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Docetaxel, erlotinib or pemetrexed is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose.

The following endpoints are measured: PK parameters, HAHA1 response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 11:

CP-675,206 in Combination with platinum-based chemotherapy for Treatment of Non-Small Cell Lung Cancer

Patients having advanced Stage MIb (with effusion) or mestatatic Stage IV NSCLC, and which have previously responded or remained stable after a platinum-based chemotherapy first line regimen, are given CP-675,206 at least about three weeks but not more than about six weeks after the lose of first line platinum-based therapy.

The patient is further administered a single IV infusion (100 mL/hr) of anti-CTLA4 antibodies as described herein at a dose of about 15 mg/kg. Prophylactic anti-emetics and anti-diarrheals are given as appropriate. The treatment is repeated after 28 days for approximately 12 cycles in the absence of intolerable toxicity or disease progression. More preferably, CP-675,206 is administered at at least about 10 mg/kg every three weeks for four cycles and then every three months thereafter.

Preferably, the patient is premedicated with antihistamine (H1) at least one half hour prior to infusion of anti-CTLA4. Premedication is recommended but not required.

Docetaxel, erlotinib or pemetrexed is administered sequentially or simultaneously with anti-CTLA4 antibody either once, or repeatedly, as determined.

CP-675,206 is provided in 20 ml clear glass vials with a rubber stopper and an aluminum seal. Each vial contains 20 mg/ml (with a nominal fill of 400 mg/vial) of CP- 675,206, in a sterile aqueous solution comprising 20 mM histidine buffer, PH 5.5, 84 mg/ml trehalose dehydrate, 0.2 mg/ml polysorbate 80, and 0.1 mg/ml disodium EDTA dehydrate.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs can be repeated post-dose, as clinically indicated. A physical examination (including ophthalmologic assessment and signs of autoimmunity) is performed on Day 1. Samples for hematology panel (hematocrit, RBC count, WBC count, differential), chemistry (Alkaline Phosphatase, calcium, chloride, GGT, LDH, magnesium, phosphorus, random glucose, sodium, urea, uric acid), urinalysis (blood, protein), others (activated partial thromboplastin time [APTT], prothrombin time (PT), autoantibody panel, C reactive protein, TSH, T3, T4, amylase, lipase, serum C3, C4, serum Ig level), are obtained. Baseline human anti-human antibody (HAHA) titer is determined and pharmacokinetic (PK) specimen is obtained pre-dose. The following endpoints are measured: PK parameters, HAHA, response rate and time to progression. Time to progression and overall survival are calculated using the Kaplan-Meier product limit method.

EXAMPLE 12:

Natural history of diarrhea associated with CTLA4 blockade by anti-CTLA4 antibody Diarrhea resulting from immune activation has been associated with CTLA4 blockade. For example in patients with stage IV melanoma receiving ipilimumab (MDX-010), a number of patients developed grade 3/4 autoimmune enterocolitis and severe diarrhea (Attia et al., 2005). In a single-dose phase I trial of CP-675,206 at doses up to 15 mg/kg in patients with solid tumors (n = 39), 9 instances of diarrhea were reported including 3 grade 3 events (Ribas et al., 2005). The incidence and severity of diarrhea was assessed in patients receiving CP-675,206 in a large phase I/I I study.

An open-label phase l/ll trial of CP-675,206 was conducted in patients with stage III (unresectable) or stage IV melanoma and an ECOG PS ≤ 1. Diarrhea was assessed in patients treated at the phase Il doses: 10 mg/kg once per month (Q1M) in phase I (n = 22), or 10 mg/kg Q1M (n = 44) or 15 mg/kg once every 3 months (Q3M) (n = 45) in phase II.

Medians of 3.5 doses (range, 1 to 18) at 10 mg/kg Q1M (10Q1M) in phase I, 3 doses (range, 1 to 26) at 10 mg/kg Q1 M in phase II, and 1 dose (range, 1 to 9) at 15 mg/kg Q3M (15Q3M) were administered with 100% dose compliance. Treatment-related diarrhea was reported by 43 (39%) of 111 patients, and grade 3 diarrhea occurred in 14 (13%) patients. One patient had grade 4 colitis resulting in a colectomy. Diarrhea (all grades) occurred with similar frequency in each dose group; however, grade 3 treatment-related diarrhea occurred in 8% of patients treated with 15 mg/kg Q3M compared with 18% of patients treated with 10 mg/kg Q1M in phase I and 14% of patients treated with 10 mg/kg Q1M in phase II. Among 9 patients with an objective response, 8 experienced diarrhea (3 of which were grade 3). The majority of cases (65%) were mild to moderate in severity with a median time to onset of 51 days (range, 1 to 583 days) and resolution of 8 days (range, 1 to 182 days). More than half of patients who reported serious events of diarrhea were treated with steroids. Diarrhea associated with CP-675,206 was primarily mild to moderate in severity, transient, and manageable. In addition, 15 mg/kg Q3M may be better tolerated than 10 mg/kg Q1 M.

EXAMPLE 13: Survival of patients with metastatic melanoma treated with anti-CTLA4 monoclonal antibody CP-675,206 in a phase l/ll study CP-675,206 has demonstrated clinical activity in patients with metastatic melanoma. Prolonged survival was observed in a prior single-dose phase I study, even in patients who did not achieve objective tumor responses.

A multidose phase l/ll trial was conducted in patients (N = 119) with histologically confirmed stage IHc (unresectable) or stage IV recurrent metastatic melanoma and ECOG PS ≤ 1. The study consisted of a phase I, open-label, multidose study (3, 6, and 10 mg/kg) and a phase I expansion cohort for HLA-A2.1 + patients (10 mg/kg monthly [10Q1 M]), followed by a phase Il open-label study of 2 dosing regimens: 10 mg/kg Q1M and 15 mg/kg every 3 months (15Q3M). The primary endpoint was safety in phase I, immune monitoring in the expansion cohort, and response in phase II. Survival was analyzed as a secondary endpoint.

In the phase I study, Kaplan-Meier estimates of median overall survival were 17.6 months for all dose groups combined (n = 28). In the phase Il study, median survival was 10.3 months in the 10 mg/kg arm and 11.0 months in the 15 mg/kg arm. Survival outcomes were favorable, compared with historical median survival of 7 months, independent of whether patients achieved an objective response.

Kaplan-Meier estimate* of median survival, months (95%

Regimen Enrolled, n Died, n (%) Cl)

Phase I 28 18 (64.3) 17.6 (8, 25.2)

3 mg/kg 3 3 (100.0) 8.0 (4.4, 17.8)

6 mg/kg 3 3 (100.0) 7.5 (4.4, 19.5)

10 mg/kg 8 6 (75.0) 19.3 (6.01 , NE)

10 mg/kg expansion 14 6 (42.9) 25.1 (9.9, NE)

Phase Il

10 mg/kg, Q1 M 44 29 (65.8) 10.3 (6.6, 13.2)

15 mg/kg, Q3M 45 32 (71.1) 11.0 (8.1 , 16.7)

*Patients censored when last known to be alive. NE = Cannot be estimated.

Patients participating in a multiple dose study of CP-675,206 showed a survival time that was greater than expected on historic controls. EXAMPLE 14:

Comparison of dose and dosing schedule of CP-675,206 anti-CTLA4 monoclonal antibody in a Phase Il clinical trial in patients with advanced melanoma A two-stage, two-arm phase Il trial was conducted to assess the optimal dosing regimen for pivotal clinical trial testing of CP-675,206.

Eligible patients had measurable melanoma (stage IUc or IV) progressing on or after prior therapy with ECOG PS ≤ 1. In stage 1 , 18 patients per arm were randomized to either 10 mg/kg once per month (10Q1M) or 15 mg/kg every 3 months (15Q3M). If 3 or more patients in either arm had complete response (CR) or partial response (PR), then 25 more patients were entered to that arm. Primary endpoint was objective tumor response (OR), and secondary endpoints were safety and survival.

Eighty-nine (89) patients received at least 1 dose (44 at 10Q1M, 45 at 15Q3M), with both study arms moving to stage 2. Ninety-six percent of patients had stage IV disease,, and 57% had elevated LDH. There were no significant differences in age, sex, stage, or baseline

LDH levels between study groups. A median of 3 doses (range, 1 to 26) at 10Q1M and 1 dose (range, 1 to 9) at 15Q3M were administered with 100% compliance. Dose delays occurred in 30% of patients treated at 10Q1M and 16% at 15Q3M. Two (2) patients at 10

Q1M and five (5) patients at 15 Q3M continued on study beyond 12 months. Six patients at 10Q1M were discontinued due to the following toxicity: three due to diarrhea/colitis (one patient requiring colectomy); one due to Grave's ophthalmopathy; one due to pancreatitis; and one due to hypersensitivity reaction. Two patients at 15Q3M were discontinued: one due to colitis and pancreatitis, and the other due to diarrhea (P = 0.14). There were no toxic deaths. The dose of 15Q3M was associated with lower incidence of grade 3 or 4 adverse events (AEs), that is, 31% compared with 41% at 10

Q1 M; P = 0.42.

Responses by investigator assessment were 1 CR and 3 PRs at 10 Q1M, and 1 CR and 2 PRs at 15 Q3M, including responses in skin, lymph nodes (LN), bone, liver, lung, and adrenal glands. Only 1 patient with PR at 10Q1 M relapsed, and the remaining responses were ongoing (18+ to 28+ months). Median survival was 10.3 months at 10Q1 M and 11.0 months at 15Q3M (P = NS).

The 15 mg/kg every three months (15Q3M) regimen was chosen for further clinical testing based on comparable antitumor efficacy and a trend to improved feasibility and safety compared with 10 mg/kg every month (10Q1M). EXAMPLE 15:

Increased anti-virus titers in combination immunization of Rhesus monkeys Combination therapy comprising administration of a vaccine and anti-CTLA4 antibody mediated an increased antiviral immune response. Rhesus macaques were immunized with FLUZONE subvirion 1998-99 formula (NDC

49281-362-11 Influenza Virus Vaccine, Trivalent Types A and B; Aventis Pasteur Inc. (includes Connaught Labs., Pasteur Merieux and Pasteur Merieux Connaught)). The 1998- 99 vaccine contained hemagglutinin A (HA) from three strains: A/Beijing/262/95 (H1N1 ), A/Sydney/5/97 (H3N2), and B/Harbin/07/94 (a B/Beijing/184/93-like strain). The monkeys were immunized intramuscularly (IM) with FLUZONE on day 0 (week 0) and again 4 weeks later (week 4). Antibody was administered according to the following schedule: Group 1 was administered anti-CTLA4 antibody CP-642,570 (5 mg/kg) intravenously (IV) on week 0; Group 2 was administered an irrelevant control antibody (anti-KLH antibody; 5 mg/kg IV) on week 0; Group 3 was immunized IM with FLUZONE on week 0 and reimmunized IM with FLUZONE on week 4 and administered anti-CTLA4 antibody (5 mg/kg IV) on week 4; Group 4 was immunized IM with FLUZONE on week 0 and reimmunized IM with FLUZONE on week 4 and administered an irrelevant control antibody (anti-KLH antibody at 5 mg/kg IV) on week 4; and Group 5 was immunized IM with FLUZONE on week 0 and reimmunized IM with FLUZONE on week 4 but no antibody was administered. The animals were followed for eight weeks after the initial immunization with FLUZONE.

The anti-CTLA4 antibody administered was human anti-human CTLA4 designated CP-642,570 (also referred to herein as antibody 4.1.1).

Serum was collected at 1 hour and 24 hours after anti-CTLA4 administration and every two weeks thereafter. The following parameters were assessed: anti-CTLA4 serum levels, IgG titers to FLUZONE, level of Neopterin, and level of 2-5 adenylate synthetase.

Plasma levels of anti-CTLA4 antibody confirmed exposure, i.e., plasma levels were approximately 75 μg/ml at 1 hour, 40-50 μg/ml at 24 hours and, for the most part, not detectable by two weeks (Figure 4). One animal in Group 3 had undetectable levels of antibody, possibly due to a missed injection or a switched tube. The animals were assessed for various markers of immune activation. Neopterin levels were variable (Figure 5). That is, an initial spike in value was detected in the anti- CTLA4 group two weeks post-immunization and treatment. Four weeks after immunization, the basal levels dropped and no differences were observed between the groups, including groups 3 and 4. Whole blood was obtained and cell pellets were collected and assayed for 2-5 adenylate synthetase. Groups 1 and 2 were compared (Figure 6). The data indicate that there was a detectable increase in the anti-CTLA4 group (Group 1 ) compared with the control group.

An increase in anti-FLUZONE IgG level was detected at six weeks in animals treated with anti-CTLA4 at the time of the second immunization (Group 3) (Figure 7). No effect on anti-FLUZONE titer was observed in any of the other groups of animals. The anti-FLUZONE IgG titers for each individual animal is depicted in Figure 8 for prebleed (week -1) (+), week 0 (open triangle), week 2 (dark triangle), week 4 (open square), week 6 (shaded circle), and week 8 (open circle). The data points are grouped according to test group, i.e., Group 1 , 2, 3, 4, and 5. Values shown in boxes indicate repeat titers from the same sample. Figure 9 shows the anti-FLUZONE serum IgG titers at week 6 in Rhesus monkeys.

Individual animal results were grouped according to test group and indicate that Group 3 animals (administered anti-CTLA4 antibody on week 4) demonstrated increased anti- FLUZONE IgG (flu-lgG) titers compared with the other animals. One animal exhibited an increased flu-lgG titer but anti-CTLA4 exposure (by plasma level assay) could not be confirmed. While some spikes in anti-flu antibody titers were observed in control groups, the titers were lower in magnitude.

In summary, an increase in flu-lgG and 2-5 adenylate synthetase was observed in animals administered anti-CTLA4 antibody suggesting that anti-CTLA4 administered to virally infected patients, to patients immunized with viral antigens, or a portion thereof, and/or when combined with anti-viral treatment enhanced therapeutic benefit.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A method for the treatment of cancer in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of CP-675,206 anti-CTLA4 antibody in combination with a therapeutically effective amount of at least one therapeutic agent, wherein said cancer and said agent are selected from the group consisting of:
(a) the cancer is non-Hodgkin's lymphoma (NHL) and wherein the agent is rituximab;
(b) the cancer is NHL and the agent is cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP);
(c) the cancer is NHL and the agent is cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (CHOP-R);
(d) the cancer is lung cancer and the agent is bevacizumab;
(e) the cancer is non-small cell lung cancer (NSCLC) and the agent is gefitinib;
(f) the cancer is NSCLC and the agent is bevacizumab;
(g) the cancer is NSCLC and the agent is a taxane and gemcitabine, and further wherein the taxane is selected from the group consisting of docetaxel and paclitaxel;
(h) the cancer is NSCLC and the agent is a taxane and a platinum compound;
(i) the cancer is NSCLC and the agent is docetaxel;
(j) the cancer is NSCLC and the agent is erlotinib;
(k) the cancer is NSCLC and the agent is pemetrexed;
(I) the cancer is NSCLC and the agent is a platinum compound;
(m) the cancer is gastric cancer and the agent is irinotecan;
(n) the cancer is gastric cancer and the agent is fluorouracil and leucovorin;
(o) the cancer is liver cancer and the agent is doxorubicin, ifosfamide and vincristine;
(p) the cancer is liver cancer and the agent is doxorubicin and vincristine;
(q) the cancer is colorectal carcinoma (CRC) and the agent is fluorouracil;
(r) the cancer is CRC and the agent is capecitabine;
(s) the cancer is CRC and the agent is fluorouracil, leucovorin, and oxaliplatin (FOLFOX);
(t) the cancer is CRC and the agent is fluorouracil, leucovorin, and irinotecan (FOLFlRI);
(u) the cancer is CRC and the agent is cetuximab;
(v) the cancer is chronic myeloid leukemia (CML) and the agent is imatinib mesylate;
(w) the cancer is chronic lymphocytic leukemia (CLL) and the agent is imatinib mesylate;
(x) the cancer is pancreatic cancer and the agent is gemcitabine; (y) the cancer is breast cancer and the agent is a taxane;
(z) the cancer is breast cancer and the agent is cyclophosphamide, doxorubicin and a taxane;
(aa) the cancer is breast cancer and the agent is selected from the group consisting of tamoxifen, anastrazole, letrozole, and fulvestrant;
(bb) the cancer is breast cancer and the agent is trastuzumab;
(cc) the cancer is breast cancer and the agent is bevacizumab;
(dd) the cancer is breast cancer and the agent is cetuximab;
(ee) the cancer is breast cancer and the agent is axitinib;
(ff) the cancer is bladder cancer and the agent is Bacillus Calmette-Guerin (BCG);
(gg) the cancer is bladder cancer and the agent is gemcitabine and cisplatin;
(hh) the cancer is melanoma and the agent is interferon alpha;
(ii) the cancer is multiple myeloma and the agent is bortezomib; βj) the cancer is multiple myeloma and the agent is dexamethasone and thalidomide; and
(kk) the cancer is ovarian cancer and the agent is carboplatin and paclitaxel.
2. The method of claim 1, wherein said treatment is selected from the group consisting of neoadjuvant therapy, adjuvant therapy, first-line therapy, second-line therapy, and third-line therapy.
3. The method of claim 1, wherein said agent is administered sequentially or contemporaneously with said antibody.
4. The method of claim 1 according to subparagraph (i), wherein said taxane is paclitaxel and wherein said platinum compound is carboplatin.
5. The method of claim 4, wherein said method further comprises administering at least one agent selected from the group consisting of bevacizumab, PF03512676, and sunitinib.
6. The method of claim 1(i), further comprising administering at least one agent selected from the group consisting of erlotinib and pemetrexed, wherein said treatment comprises second line therapy.
7. The method of claim 1 , wherein said therapeutically effective amount of said antibody, or portion thereof, ranges from about 1 mg/kg to 40 mg/kg.
8. The method of claim 7, wherein said amount ranges from about 3 mg/kg to 15 mg/kg.
9. The method of claim 1 according to subparagraph (s), wherein said treatment is selected from the group consisting of first line therapy and adjuvant therapy following surgical resection of a primary colon tumor.
10. The method of claim 1 according to subparagraph (x), wherein said pancreatic cancer is selected from the group consisting of non-resectable Stage II, locally advanced Stage III, and metastatic Stage IV and wherein said treatment comprises first line therapy.
11. The method of claim 1 according to subparagraph (x), wherein said antibody is administered after administration of the last dose of gemcitabine.
12. The method of claim 17, wherein said antibody is administered at least about three weeks after and within at least six weeks of administration of the last dose of gemcitabine.
13. The method of claim 1 according to subparagraph (y), wherein said treatment comprises first line therapy.
14. The method of claim 1 according to subparagraph (kk), wherein said treatment comprises first line therapy.
15. The method of claim 1 according to subparagraph (I), wherein said NSCLC responded or remained stable after about six cycles of administration of said platinum compound and further wherein said antibody is administered after said platinum-based therapy.
16. A method for preventing or treating infection by HIV or for preventing, treating or delaying the onset of AIDS in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of anti-CTLA4 antibody CP- 675,206 and further comprising administering a therapeutically effective amount of at least one antiviral agent selected from the group consisting of an HIV protease inhibitor, a non- nucleoside reverse transcriptase inhibitor, a nucleoside/nucleotide reverse transcriptase inhibitor, a CCR5 antagonist, an inhibitor of gp120 interaction with CD4, an HIV fusion inhibitor, a HIV integrase inhibitor, an RNaseH inhibitor, a prenylation inhibitor, and a maturation inhibitor.
17. The method of claim 16, wherein said CCR5 antagonist is maraviroc.
18. The method of claim 17, said method further comprising assessing the co- receptor tropism of said HIV.
19. A method for preventing or treating infection by HIV or for preventing, treating or delaying the onset of AIDS in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of an anti-CTLA4 antibody and maraviroc.
20. The method of claim 19, wherein said anti-CTLA4 antibody is selected from the group consisting of CP-675,206 and ipilimumab.
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