BRPI0515735A2 - combination treatment of ctla4 antibody and aromatase inhibitor for breast cancer, their pharmaceutical compositions and their uses - Google Patents

Abstract

COMBINATION TREATMENT OF ANTI-BODY OF CTLA4 AND AROMATASE INHIBITOR FOR BREAST CANCER, ITS PHARMACEUTICAL COMPOSITIONS AND THEIR USES. The invention relates to the administration of an anti-CTLA4 antibody, particularly human antibodies to human CTLA4, such as those having antibody amino acid sequences 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, and 10D1 (MDX-01O), in combination with an aromatase inhibitor, for the treatment of breast cancer . More particularly, the invention relates to the administration of an anti-CTLA4 antibody and exemestane for the treatment of breast cancer.

Description

Patent Descriptive Report for "CTLA4 ANTIBODY COMBINATION TREATMENT AND ARO MATASE INHIBITOR FOR BREAST CANCER, ITS PHARMACEUTICAL COMPOSITIONS, AND THEIR USES".

Background of the Invention

Each year, more than 180,000 women are diagnosed with breast cancer in the United States. If current breast cancer rates remain constant, every woman born today has one in ten chances of developing breast cancer at a median age of about 60 to 65.

Approximately two thirds of postmenopausal breast cancer patients have estrogen-dependent disease thus making them treatable by anti-estrogen therapy. Estrogens promote growth and proliferation of specific target cells, such as estrogen-dependent breast carcinoma cells and breast epithelium (Brueggemeier Amer J Therapeutics 8: 333-344 (2001)). Thus, systemic adjunctive treatment therapy administered in addition to the main treatment of localized breast cancer (eg, breast and radiation conservation surgery or mastectomy) focused on agents that block the estrogen receptor in tumor cells. Such antihormonal agents include tamoxifen (NOVALDEX D, SOLTAMOX, TAMOFEN) which inhibits estrogen binding with the estrogen receptor, fulvestrant (FASLODEX), which degrades the receptor, as well as aromatase inhibitors (eg anastrozole (ARIMIDEX) , Ietrozole (FEMARA), and exemestane (AROMASIN)), which blocks the androgen aromatase enzyme catalysis to estrogens, thereby decreasing the amount of hormone ligand available to bind with the receptor.

Despite advances in hormone therapies or other breast cancer therapies, it is estimated that 40,921 women will die of advanced or recurrent breast cancer in 2004 (American Cancer Society, Statistical Cancer (2004)). There is currently no curative treatment available for patients with lasting disease and therapy is essentially palliative. Thus, new therapies, such as combination of anti-hormonal agent with immunotherapy, are important in the treatment of breast cancer. More specifically, a cytotoxic T lymphocyte-associated antigen 4 cancer immunotherapy approach targets 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), releases 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 Immunol. & Cell Biol. 77: 1-10 (1999)). Several studies have shown that CTLA4 blockade employing antibodies markedly enhances T-cell mediated death of tumors and can induce antitumor immunity (Leach et al., Science 271: 1734-1736 (1996); Kwon et al. Proc. Natl Acad. Sci. USA 94: 8099-8103 (1997); Kwon et al., Natl. Acad. Sci. USA 96: 15074-15079 (1999)).

Although the use of anti-CTLA4 antibodies to induce an antitumor response holds great promise in cancer treatment, and despite the promise of anti-hormone therapies in breast cancer treatment, there is a need to develop new therapies to treat breast cancer. mama. The present invention satisfies this need.

Summary of the Invention

The invention includes a method for treating breast 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, or antigen binding portion thereof, in combination with a therapeutically effective amount of an α-romatase inhibitor.

In one aspect, the aromatase inhibitor is at least one inhibitor selected from the group consisting of anastrozole, Ietrozole, and example. In another aspect, the aromatase inhibitor is exemestane.

In one aspect, the therapeutically effective amount of an amount of human anti-CTLA4 antibody ranges from about 1 mg / kg to 40 mg / kg. In another aspect, the therapeutically effective amount of an amount of human anti-CTLA4 antibody ranges from about 3 mg / kg to 15 mg / kg.

In yet another aspect, the therapeutically effective amount of exemestane ranges from about 25 mg per day to 200 mg per day.

In another aspect, the therapeutically effective amount of exemestane is about 25 mg per day.

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

In another aspect, the antibody is selected from a group consisting of a non-human mammalian antibody, a chimeric antibody, and a human antibody.

In one aspect, the antibody is a human anti-CTLA4 antibody.

In another aspect, the anti-CTLA4 antibody, or antigen binding portion thereof, is at least one antibody selected from the group consisting of:

(a) a human antibody that has a CTLA4 binding affinity of about 10-8 or more, and which inhibits binding between CTLA4 and

B7-1, and binding between CTLA4 and B7-2;

(b) a human antibody having an amino acid sequence comprising at least one human CDR sequence corresponding to a CDR sequence 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1;

(c) a human 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, 11.6.1, 11.7.1., 12.3.1.1, and 12.9.1.1;

(d) a human antibody having the amino acid sequences of a heavy chain variable region and a light chain variable region 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1;

(e) an antibody, or an antigen binding portion thereof, that competes for CTLA4 binding with at least one 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1; and

(f) an antibody, or antigen binding portion thereof, which interacts for CTLA4 binding with at least one 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1.

In another aspect, the antibody is a human antibody that has antibody heavy and light chain amino acid sequences 11.2.1.

In yet another aspect, the antibody comprises a heavy chain and a light chain wherein the amino acid sequences of the heavy chain heavy chain variable region and the light chain variable region are selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 9;

(b) the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 21;

(c) the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 33;

(d) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 1 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 7;

(e) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 13 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 19;

(f) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 25 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 31;

(g) the amino acid sequence of a heavy chain variable region and a 10D1 antibody light chain variable region.

In one aspect, the antibody, or antigen binding portion thereof, is an antibody selected from the group consisting of:

(a) an antibody that has a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and also having a chain variable region. light comprising the amino acid sequences set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12;

(b) an antibody having a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and also having a light chain variable region which comprises the amino acid sequences shown SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24;

(c) an antibody having a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and also having a light chain variable region which comprises the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; and

(d) an antibody having a heavy chain variable region comprising the 10D1 antibody heavy chain amino acid sequences CDR1, CDR2, and CDR3, also having a light chain variable region comprising the CDR1 light chain amino acid sequences , CDR2, and 10D1 antibody CDR3.

In another aspect, the method also comprises administering to the patient at least one agent selected from the group consisting of an alkylating agent, a folate antagonist, a pyrimidine antagonist, an anthracycline antibiotic, a platinum combination, a taxane, a vinca alkaloid, a camptothecin analog, a toll-like receptor stimulating agent, a heat shock protein-based tumor vaccine, an antigen-presenting cell-based therapy, a mammalian target of rapamycin inhibitor, an erbB2 inhibitor, an EG-FR1 inhibitor a VEGF1 inhibitor a VEGFR inhibitor, an angiogenic inhibitor, an antibody, an immunomodulator, a selective estrogen receptor modulator, a cytokine, a tumor vaccine, antiproliferative agent, an immune costimulatory molecule, and a cytokine.

The invention includes a pharmaceutical composition for the treatment of breast cancer comprising: a therapeutically effective amount of an anti-CTLA4 antibody; a therapeutically effective amount of an aromatase inhibitor; and a pharmaceutically acceptable carrier.

In one aspect, the aromatase inhibitor is at least one aromatase inhibitor selected from the group consisting of anastrozole, letrozole, and exemestane.

In another aspect, the aromatase inhibitor is exemestane. The invention includes a use of an amount of an anti-CTLA4 antibody in the preparation of a breast cancer treatment composition in a patient wherein the treatment also comprises administering to the patient an amount of an aromatase inhibitor.

In one aspect, the aromatase inhibitor is exemestane and also where the antibody is a human antibody having antibody heavy and light chain variable region amino acid sequences 11.2.1. 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 accompanying drawings. In order to illustrate the invention, embodiments which are currently preferred are shown in the drawings. 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 for anti-CTLA4 antibody 4.1.1. Figure 1A shows the life-size nucleotide sequence for heavy chain 4.1.1 (SEQ ID NO: 1). Figure 1B shows the full-length amino acid sequence for the heavy chain 4.1.1 (SEQ ID NO: 2), and the amino acid sequence for the heavy chain variable region 4.1.1 (SEQ ID NO: 3) designated in square 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: VIWYDGRNKY-YADSV (SEQ ID NO: 5); and CDR3: GGHFGPFDY (SEQ ID NO: 6). Figure 1C shows the nucleotide sequence for light chain 4.1.1 (SEQ ID NO: 7). Figure 1D shows the life-size light chain amino acid sequence 4.1.1 (SEQ ID NO: 8), and the variable region as indicated in 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: CQQYGTSPWT (SEQ ID NO: 12).

Figure 2, comprising Figures 2A-2D, shows the nucleotide and amino acid sequences for anti-CTLA4 antibody 4.13.1. Figure 2A shows the full-length nucleotide sequence for the heavy chain 4.13.1 (SEQ ID NO: 13). Figure 2B shows the full-length amino acid sequence for the heavy chain 4.13.1 (SEQ ID NO: 14), and the amino acid sequence for the heavy chain variable region 4.13.1 (SEQ ID NO: 15) designated between the square 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: VIWYD-GRNKDYADSV (SEQ ID NO: 12); and CDR3: VAPLGPLDY (SEQ ID NO: 18). Figure 2C shows the nucleotide sequence for light chain 4.13.1 (SEQ ID NO: 19). Figure 2D shows the amino acid sequence of the life-size light chain 4.13.1 (SEQ ID NO: 20), and the variable region as indicated in 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: CQQYGRSPFT (SEQ ID NO: 24).

Figure 3, comprising Figures 3A-3D, shows the nucleotide and amino acid sequences for anti-CTLA4 antibody 11.2.1. Figure 3A shows the life-size nucleotide sequence for heavy chain 11.2.1 (SEQ ID NO: 25). Figure 3B shows the full-length heavy chain amino acid sequence 11.2.1 (SEQ ID NO: 26), and the amino acid sequence for the heavy chain variable region 11.2.1 (SEQ ID NO: 27) designated between the brackets "[]". The amino acid sequence of each 11.2.1 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 light chain 11.2.1 (SEQ ID NO: 31). Figure 3D shows the life-size light chain amino acid sequence 11.2.1 (SEQ ID NO: 32), and the variable region as indicated in 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).

Detailed Description of the Invention

The invention relates to anti-CTLA4 antibodies employed in combination with at least one aromatase inhibitor, for example, anastrozole, letrozole, and exemestane, to treat breast cancer in a patient in need of such treatment. The invention also relates to the treatment of breast cancer by combining the antibody-aromatase inhibitor combination with another agent or agents.

Unless otherwise defined herein, the scientific and technical terms employed in connection with the present invention will have the meanings that are generally understood by those skilled in the art. In addition, unless otherwise required by context, singular terms will include pluralities and plural terms will include singular. Generally, the nomenclatures employed with respect to and techniques of tissue and cell culture, molecular biology, immunology, microbiology, genetics and hybridization, and nucleic acid and protein chemical described herein are those well known and generally employed in the art. technique. 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 which are cited and described throughout this specification unless otherwise indicated. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), Ausubel and the like, 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 generally performed in the art or as described herein. The nomenclatures employed with respect to, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and generally employed in the art. Standard techniques are employed for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and release, and treatment of patients.

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

Articles "one" and "one" are used herein to refer to one or more than one (i.e. at least one) of the article's grammatical object. By way of example, "one 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 Synthe-sis (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 replaced by another amino acid residue that has an R chain 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 upward 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, for example, Pearson, Methods Mol. Biol. 243: 307-31 (1994).

Examples of amino acid groups having 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) acid side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are valine leucine isoleucine, phenylalanine tyrosine, lysine arginine, alanine valine, glutamate aspartate, and asparagine glutamine.

Alternatively, a conservative substitution is any change that has a positive value in the log probability matrix PAM250 described in Gonnet et al., Science 256: 1443-45 (1992), incorporated herein by reference. A "moderately conservative" substitution is any change having a nonnegative value in the PAM250 log probability matrix.

Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, and (4) confer or modify other functional or physicochemical properties of such analogs. Analogues comprising substitutions, deletions, and / or insertions may include multiple muteins of a sequence except 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 at the portion of the polypeptide outside the domain (s) forming intermolecular contacts, for example outside the CDRs). A conservative amino acid substitution should not substantially alter the structural characteristics of the parent sequence (for example, a replacement amino acid should not serve to break a helix that occurs in the parent sequence, or to break other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, S-tructures 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 and the like, 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 combines similar sequences employing similarity measures designed for various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, 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 protein and nucleotide sequences. GCG contains programs such as "Gap" and "Bestfit" that can be employed with default parameters to determine sequence similarity, homology and / or sequence identity between closely related polypeptides, such as homologous polypeptides of different species of organisms or between a wild type protein and a mutein thereof. See, for example, GCG version 6.1, version 9.1, and version 10.0.

Polypeptide sequences can also be compared using FASTA using the recommended or default parameters, a program in GCG Version 6.1. FASTA (eg, FASTA2 and FAS-TA3) provides alignments and sequence identity percentages of the regions of best overlap between search and query sequences and (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson , Me-thods Mol. Biol. 132: 185-219 (2000)). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially blastp or tblastn, employing default parameters. See, for example, Altschul and the like, J. Mol. Biol. 215: 403-410 (1990); Altschul and the like, Nucleic Acids Res. 25: 3389-402 (1997); hereby incorporated by reference.

An "aromatase", when the term is used herein, refers to an enzyme (e.g., 450arOm) that catalyzes the aromatization of ring A from an androgen substrate (mainly androstenedione) to estrone.

"Aromatase inhibitor" means a compound or substance which when contacted with an aromatase detectably inhibits ring formation of an androgen substrate compared to the level of such aromatization in the absence of the compound or substance. Any assay to assess the level of enzymatic activity by an aromatase may be employed to assess inhibition and / or aromatase level. An aromatase inhibitor includes, but is not limited to, a steroid and non-steroidal inhibitor, among other things, exemestane, anastrozole, letrozole, fadrozole, vorozole, and formestane, and the term encompasses any aromatase inhibitor either now known or identified in the future.

An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Generally see, 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 a domain, Cl-Vh and Vl regions can also be subdivided into hyper-variability regions, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl termination in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid designation for each domain is according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia & Lesk1 J. MoL 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 life-size antibody. Examples of binding fragments encompassed by the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, Cl and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the Vh and CH1 domains; (iv) an Fv fragment consisting of the single-arm Vh and Vl domains of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) an isolated complementarity determining region (CDR). In addition, although the two Fv fragment domains, V1 and VH, are encoded for separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein strand in which they are linked. regions of V1 and Vh join to form monovalent molecules (known as single Fv chain (scFv)); see for example, Bird and the like Science 242: 423-426 (1988) and Huston and the like Proc. Natl. Acad. Know. 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 the Vh and Vl domains are expressed in a single polypeptide chain, but employing a linker that is too short to allow union between the two domains in the same chain, thereby forcing the domains to unite with the complementary domains of another chain and create two antigen binding sites (see, for example, Holliger and the like. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak and the like Structure 2 : 1121-1123 (1994)).

In addition, an antibody or antigen binding portion thereof may be part of larger immunoadhesion molecules formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin nucleus region to prepare a tetrameric scFv molecule (Kipriyanov and the like Human Antibodies and Hybridomas 6: 93-101 (1995)) and use of a cysteine residue, a marker peptide and a C-terminal polystidine label to prepare biotinylated bivalent scFv molecules (Kipriyanov et al. Mol. Immunol. 31: 1047-1058 (1994)). Other examples include where one or more CDRs of an antibody are incorporated into a molecule either covalently or non-covalently to make it an irpunoadhesin 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 non-covalently incorporated. Antibody moieties, such as Fab and F (ab) 2 fragments, may be prepared from whole antibodies employing conventional techniques, such as pepsin or papain digestion, respectively, from whole antibodies. In addition, antibodies, antibody moieties and immunoadhesion molecules can be obtained by employing 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 be employed herein as well. An antigen binding portion competes with the intact antibody for specific binding. Generally see, Fundamental Immunolog, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). Antigen binding moieties may be produced by recombinant DNA techniques or by chemical or enzymatic cleavage of intact antibodies. In some embodiments, antigen binding moieties 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 grant 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", when used interchangeably herein, include antibodies that have constant and variable regions (if present) derived from human germline immunoglobulin sequences. Human sequence antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by specific or random site 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 germline-derived CDR sequences from another mammalian species, such as a mouse, have been grafted onto human framework sequences ( that is, "humanized" or PRI-MATIZED ™ antibodies).

The term "chimeric antibody" as used herein means an antibody comprising regions of 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 CDRs are derived from a human anti-CTLA4 antibody. In another embodiment, CDRs of more than one human anti-CTLA4 antibody are combined into one chimeric human antibody. For example, a chimeric antibody may include a light chain CDR1 of a first anti-human CD40 antibody, a light chain C-DR2 of a second human anti-CTLA4 antibody, and a light chain CDR3 and CDR3 of a third anti-human antibody. -CTLA4, and heavy chain CDRs may be derived from one or more other anti-CD40 antibodies. In addition, framework regions may be derived from one of the same anti-CTLA4 antibodies or one or more different humans.

In addition, as described hereinabove, the chimeric antibody includes an antibody comprising a portion derived from germline sequences of more than one species.

"Glycoform" refers to a complex oligosaccharide structure comprising bonds of various carbohydrate moieties. Such structures are described in, for example, 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, for example, International Patent Publication No. WO 99/22764).

"Glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently linked to a protein (e.g., glycoform) as well as sites to which glycoform (s) are covalently linked to a protein's peptide backbone , more specifically to an immunoglobulin protein.

Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation and / or glycoform patterns compared to 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 present invention, independent of antibody glycosylation.

By the term "effective amount" or "therapeutically effective amount" as used herein is meant an amount which 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, for example. such methods as described herein.

The skilled artisan would understand that the effective amount of the compound or composition administered herein varies and can easily be determined based on various 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 "competes" as used herein with respect to an antibody, it is understood that a first antibody, or an antigen binding portion thereof, competes for binding with a second antibody, or an antigen binding portion thereof, where The binding of the first antibody to 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, may, however, not necessarily be the case. That is, a first antibody may inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of the other antibody to its epitope or cognate ligand, whether to an equal, greater or lesser extent, the antibodies are said to "cross compete with each other for binding of their respective epitopes. For example, cross-competing antibodies may bind to the epitope, or epitope potion, to which the antibodies employed in the invention bind.The use of both competition and cross-competition antibodies is encompassed by the present invention. of the mechanism by which such competition or cross-competition occurs (for example, steric hindrance, conformational change, or binding to a common epitope, or portion thereof, and the like), the skilled artisan would appreciate, based on the teachings provided herein, that such competition and interaction antibodies were encompassed and could be useful for the methods described herein.

The term "epitope" includes any protein determinant capable of specific binding to a T cell receptor or immunoglobulin. Epitopic determinants usually consist of chemically active surface groupings of molecules such as sugar side chains or amino acids and usually have three specific dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished by the fact that binding to the former but not the latter is lost in the presence of denaturing solvents. "Instructional Material," when such term is used herein, includes

a publication, record, diagram, or other means of expression that may be employed to communicate the utility 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 alternatively, the instructional material may describe one or more methods for alleviating disease or disorder in a cell, tissue, or mammal, including as described elsewhere herein.

The instructional material of the kit may, for example, be attached to a container containing the compound and / or composition of the invention or may be carried along with a container containing the compound and / or composition. Alternatively, the instructional material may be transported separately from the container with the intention that the container uses the instructional material and the compound cooperatively.

Except when noted, the terms "patient" or "individual" are used interchangeably and refer to mammals such as human patients and nonhuman primates, as well as veterinary individuals such as rabbits, rats, and mice, and the like animals. Preferably, patient refers to a human.

Conventional observation is employed here to portray polypeptide sequences: the left end of a polypeptide sequence is the amino terminus; The right end of a polypeptide sequence is the carboxyl terminus.

By the phrase "specifically binds," as used herein is meant a compound, for example, a protein, a nucleic acid, an antibody, and the like that recognizes and binds a specific molecule, but does not substantially recognize or bind other molecules. in a sample. For example, an antibody or peptide inhibitor that recognizes and binds a cognate ligand (for example, 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 the designated assay conditions, the specified binding moiety (e.g., an antibody or antigen binding moiety thereof) preferentially binds 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 employed to select an antibody that specifically binds a molecule of interest. For example, solid phase ELISA immunoassay, immunoprecipitation, Western blot analysis and BIAcore are employed to identify an antibody that specifically reacts with CTLA4. Typically a specific or selective reaction will be at least twice the base signal and noise and more typically more than 10 times the base, even more specifically, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (K0 ) is <1 μΜ, preferably <100 nM and preferably <10 nM. The term "Kd" refers to an equilibrium dissociation constant of a particular antibody-antigen interaction.

As used herein, "substantially pure" means an object species which is the predominant species present (that is, on a molar basis is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition where object species (for example, 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%. More preferably, the object species is purified for essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) where the composition consists essentially of a single macromolecular species.

As used herein, "treating" means reducing the frequency with which symptoms of a disease (ie, metastasis and / or tumor growth, or other effect mediated by activity and / or immune cell numbers, and the like) are experienced. by a patient. The term includes administering the compounds or agents of the present invention to prevent or delay the onset of symptoms, complications, or biochemical indications of a disease (e.g., elevation of PSA level), alleviate symptoms or also arrest or inhibit development. of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the onset of clinical or subclinical symptoms) or therapeutic suppression or relief of symptoms after the onset of the disease.

I. Aromatase Antibody-Inhibitor Combination Therapy

The invention relates to methods for the administration of a human CTLA4 binding antibody, or an antigen binding portion of the antibody, in combination with hormone therapy (e.g. aromatase inhibitor) to treat breast cancer. The combination of C-TLA4 blockade with reduction in estrogen level in a patient according to a method of the invention may provide a synergistic therapeutic benefit as more fully described below.

Although the invention encompasses numerous combination therapies wherein the antibody is administered to the patient in combination with at least one aromatase inhibitor, the present invention is by no means limited to the exemplified agents, which are presented herein for illustrative purposes only.

In one embodiment, a combination of a C-TLA4 antibody, or an antigen binding portion thereof, and an aromatase inhibitor ("Al") is administered to a breast cancer patient. In one aspect, the aromatase inhibitor is selected from the group consisting of anastrozole, ietrozole and exemestane which are commercially available as ARIMIDEX, FEMARA and AROMAS IN respectively. More preferably, the aromatase inhibitor is exemestane. This is because a synergistic or additive effect is mediated by administration of a combination comprising an anti-CTLA4 antibody and an Al. Without wishing to be bound by any particular theory of the invention, Al therapy may be increased, perhaps by apoptotic or other changes. architectural changes, exposure of breast cancer-specific tumor antigen (s) to the immune system such that the immune response to the tumor cell is increased. That is, hormone therapy can create or augment a specific tumor antigen source mediated by tumor cell death that can successively feed tumor antigen into host antigen presenting pathways. The anti-CTLA4 antibody mediates an enhanced immune response to increased tumor specific antigen levels in the antigen presentation pathway thereby providing a potential synergistic therapeutic effect when combined with aromatase inhibitor therapy. Other combination therapies that may result in anti-CTLA4 enhancement synergy of the immune response through the release of cell death from specific tumor antigens are radiation, surgery, and chemotherapy, among others. Without wishing to be bound by any theory of the invention, the combination of CTLA4 blockade and aromatase inhibition may induce a more robust immune response to breast cancer than expected. Therefore, the combination of aromatase blockade employing, among others, exemestane, in combination with an anti-CLTA4 antibody, can provide a potential synergistic effect thereby providing an important new therapeutic treatment for breast cancer.

The methods of the invention may be performed as a neoadjuvant therapy prior to surgery, radiation therapy, or any other localized treatment in order to sensitize tumor cells or otherwise grant a therapeutic benefit to the patient. However, the invention is not limited to the neoadjuvant composition. Preferably, the methods of the invention may be employed throughout the entire disease and continuum treatment, for example, but not limited to, adjuvant therapy, first line, second line, third line, and the like, for breast cancer.

In one embodiment, breast cancer is estrogen receptor positive (ER +), such that aromatase inhibition reduces the estrogen level thereby affecting the tumor. In one aspect, the ER + tumor is present in a postmenopausal (postmenopausal) female patient, such that a decrease in estrogen level mediates a therapeutic antitumor effect on the patient. However, while it may be preferable to administer Al therapy to a postmenopausal ER + patient, the present invention is not limited to such a patient, but preferably comprises administration of anti-CTLA4 aromatase inhibitor-antibody therapy. to any patient who may derive a therapeutic benefit from a combination of decreased estrogen level combined with CTLA4 blockade immune enhancement, as determined by the skilled artisan equipped with the teachings provided herein.

II. Dosing Regimens

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

Non-limiting ranges, exemplary for a therapeutically effective amount of an antibody administered according to the invention, are at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg. 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 should 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 should also be understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual's need and professional judgment of the person administering or supervising the administration of the compositions, and that The dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Appropriate dosages and determinant regimens for administration of the antibody are well known in the relevant art and would be understood to be within the skill of the art as provided with the teachings described herein.

In one embodiment, the antibody is administered in an intravenous formulation as a sterile aqueous solution containing about 5 to 20 mg / ml antibody in an appropriate buffer system.

In one embodiment, for low dose administration, part of the dose is administered by an intravenous bolus and the remainder by infusion of the antibody formulation. For example, an intravenous injection of 0.01 mg / kg of antibody may be given as a bolus, and the remainder of a predetermined antibody dose may be administered by intravenous injection. In another embodiment, the whole low dose is administered as a single bolus injection. At higher doses, eg 3 mg / kg, the antibody is not administered as a bolus, but the total amount is administered by infusion. A predetermined dose of the antibody may be administered, for example, over a period of about one and a half hours to about five hours.

The present invention relates to the administration of a combination of an anti-CTLA4 antibody and an aromatase inhibitor. The skilled artisan would appreciate that the combination could be administered simultaneously or the two agents could be administered at different times. For example, in one embodiment, the antibody is administered as a single infusion and / or injection and the aromatase inhibitor (e.g., e-xemestate) is administered once daily starting before, during, or after administration of the antibody. . Typically, exemestane is given daily, once a day, usually at the same time of day and accompanied with a meal. However, the present invention is not limited to any particular dosage or administration regimen for an aromatase inhibitor. Preferably, the optimal dose, routine and regimen for administration of the antibody and aromatase inhibitor may be readily determined by one of ordinary skill in the art using well-known methods. For example, a single dose or multiple doses of the antibody may be administered. Alternatively, at least one dose, or at least three, six or 12 doses may be administered. Doses may be administered, for example, every two 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 annually. In addition, the aromatase inhibitor may be administered daily, several times or once a day, weekly, every two weeks, every third week, every fourth week, monthly, every three months, every six months, once a year. , or any other period that provides a therapeutic benefit to the patient as determined by the qualified physician.

The antibody may be administered until disease progression, or intolerable toxicity, or up to 12 successive cycles, regardless of the shortest time. The antibody may also be administered employing a regimen comprising administering a loading dose followed by a lower one. Repeated courses of at least one, and more preferably several rounds of antibody and an aromatase inhibitor may be administered to a patient experiencing a tumor recurrence which has previously had benefit derived from administration of the combination of an anti-CTLA4 antibody and a aromatase inhibitor.

In one embodiment, a single injection comprising the anti-CTLA4 antibody is administered to a patient intravenously at a dose of about 3 mg / kg every twenty eight days. A dose of approximately 25 mg per day of exemestane is also administered to the patient, or prior to, during and / or after administration of the antibody. In one embodiment of the invention, a single dose comprising the 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 months. A dose of approximately 25 mg per day of exemestane is also administered to the patient, or prior to, during and / or after administration of the antibody. In another embodiment, a bolus injection comprising an anti-CTLA4 antibody is administered to a patient intravenously at a dose higher than 3 mg / kg (e.g., 6, 10, 15, 20, 25, 30, 35 or 40 mg / kg) every twenty-eight days. The dose may be adjusted as known in the art based on, among similar factors, toxicity, if any, and therapeutic efficacy. A dose of approximately 25 mg per day of exemestane is also administered to the patient, or prior to, during and / or after administration of the antibody.

The aromatase inhibitor-antibody combination may be administered as a first-line systemic adjuvant therapy, or may be administered to the patient as a neoadjuvant therapy prior to surgery, radiation therapy, or any other treatment to sensitize the conditions. tumor cells or otherwise grant a therapeutic benefit to the patient.

In addition, the combination may be administered as a

second-line therapy, such as, but not limited to, once first-line tamoxifen therapy has failed. Alternatively, the combination may be administered concurrently with tamoxifen therapy, and or at any time during tamoxifen therapy, which is typically administered for about five years following initial treatment. In another embodiment, the combination of anti-CTLA4 antibody and exemestane may be administered to a next patient about two or three years of tamoxifen or other adjuvant therapy. This is because it has been shown that following exemestane therapy for about two to three years of tamoxifen therapy provides a therapeutic benefit while reducing the side effects of tamoxifen therapy.

Thus, a combination of an anti-CLTA4 antibody and an aromatase inhibitor, preferably exemestane, may provide a therapeutic benefit once tamoxifen therapy has failed, since systemic adjuvant therapy employing another a-romatase inhibitor (eg, anastrozole, letrozole, and the like) have failed, and / or after tamoxifen administration for about two to three years, among the like. Therefore, the invention encompasses the administration of an anti-CTLA4 antibody and aromatase inhibitor in combination with or following additional anti-hormone therapy, including, but not limited to, tamoxifen, anastrozole, etrozole and fulvestrant as would be appreciated by one. skilled in the art based on the description provided herein. III. Anti-CTLA4 Antibodies

In one embodiment, the CTLA4 antibody employed in the invention comprises a heavy chain wherein the Vh amino acid sequence comprises the amino acid sequences set forth in SEQ ID NOs: 3, 15 and 27. In yet another embodiment, the CTLA4 antibody V1 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. More preferably, the antibody is ticilimumab (also known as CP-675,206) which has antibody heavy and light chain amino acid sequences 11.2.1.

In yet another embodiment, the heavy chain amino acid sequence 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 nucleic acid encoded amino acid sequences comprising the sequences of nucleic acid shown 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 shown 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 shown in SEQ ID NO: 25 (heavy chain 11.2.1) and SEQ ID NO: 31 (light chain 11.2.1), respectively.

In addition, the antibody may comprise a heavy chain amino acid sequence comprising human CDR amino acid sequences derived from the 3-30 or 3-33 Vh gene, or conservative substitutions or somatic mutations herein. It is understood that the Vh 3-30 or Vh 3-33 gene encodes from FR1 to 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%, even more preferably at least 91%, even more preferably at least 94%, even more preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and preferably 100% identity, with the sequence FR1 through FR3 of a selected antibody of 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, 11.6.1, 11.7.1, 12.3.1.1, 2.9.1.1 , 10D1, and DP-50.

The antibody may also comprise the CDR regions in its light chain derived from the A27 or 012 gene or 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, 11.6.1, 11.7.1, 12.3.1.1, 2.9.1.1, 10D1.

In other embodiments of the invention, the antibody inhibits the binding between CTLA4 and B7-1, B7-2, or both. Preferably, the antibody may inhibit B7-1 binding with an IC 50 of about 100 nM or less, more preferably about 10 nM or less, for example about 5 nM or less, even more preferably about 2 nM. nM or less, or even more preferably, for example, about 1 nM or less. Likewise, the antibody may inhibit binding to B7-2 with an IC50 of about 100 nM or less, more preferably 10 nM or less, for example, even more preferably about 5 nM or less, even more preferably. about 2 nM or less, or even more preferably about 1 nM or less.

Also, in another embodiment, the anti-CTLA4 antibody has a CTLA4 binding affinity of about 10-8, or higher affinity, more preferably about 1CT9 or higher affinity, more preferably, about 10 ~ 10. or higher affinity, and even more preferably about 10 ~ 11 or higher affinity.

The anti-CTLA4 antibody includes an antibody that competes for binding to an antibody that has heavy and light chain amino acid sequences from an antibody selected from the group consisting of 4.1.1, 6.1.1, 11.2.1, 4.13. 1 and 4.14.3. In addition, anti-CTLA4 antibody may compete for binding with 10D1 antibody.

In another embodiment, the antibody preferably interacts with an antibody that has a heavy and light chain sequence, a variable and a light heavy chain sequence, and / or antibody heavy and light CDR sequences 4.1.1, 4.13. 1, 4.14.3, 6.1.1. or 11.2.1. For example, the antibody may bind to the epitope to which an antibody having heavy and light chain amino acid sequences, variable sequences and / or CDR sequences, from an antibody selected from the group consisting of 4.1.1, 4.13 .1, 4.14.3, 6.1.1, or 11.2.1 turns on. In another embodiment, the antibody interacts with an antibody that has 10D1 heavy and light chain sequences or antigen binding sequences (MDX-D010).

In another embodiment, the invention is practiced employing an anti-CTLA4 antibody comprising 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-1, CDR-2, and CDR-3, from 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, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1, or sequences having alterations of the CDR sequences selected from the group consisting of conservative changes, where conservative changes are selected from the group consisting of substitution non-polar waste by other non-polar waste, replacement of polar-loaded waste with other polar non-charged waste, replacement of polar-loaded waste with other polar-charged waste, and replacement of structurally similar waste; non-conservative substitutions, where non-conservative substitutions are selected from the group consisting of polar charged residue substitution for polar uncharged residues and non-polar residue substitution for polar residues, additions and deletions.

In another embodiment of the invention, the antibody contains less than 10, 7, 5, or 3 germline sequence amino acid changes in the CDR structure or regions. In another embodiment, the antibody contains less than 5 amino acid changes in framework regions and less than changes in CDR regions. In a preferred embodiment, the antibody contains less than 3 amino acid changes in the framework regions and less than 7 changes in the CDR regions. In a preferred embodiment, changes in framework regions are conservative and those in 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%, even more preferably at least 94%, preferably at least 95%, more preferably at least 99% of sequence identity (eg amino acid, nucleic acid, or both) or sequence similarity over the full-length light and heavy chain sequences, or the heavy or light chain separately with the antibody sequences 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1 , 10D1. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy chain and light chain, or with the heavy chain or light chain, separately from 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, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, 10D1.

In another embodiment, the antibody shares at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, more preferably at least 95%, even more preferably. at least 99% of sequence identity or sequence similarity over the light and heavy chain life-size sequences, or the light or heavy chain separately, with the Vk A27 germline, Vk 012 germline sequences, and germ line 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 DP50 germline heavy chain sequence and / or the A27 germline light chain sequence, or germline 012.

In one embodiment, the antibody shares 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, preferably at least 95%, more preferably at least 99%. , sequence identity (eg amino acid, nucleic acid, or both) or sequence similarity over the light and heavy chain variable region sequences, or the light or heavy chain variable region sequence separately, with the antibody sequences 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9 1.1, 10D1. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the light chain and heavy chain variable region sequences, or with the light chain or heavy 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, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, 10D1.

In another embodiment, the antibody shares at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, more preferably at least 95%, even more preferably. at least 99% of sequence identity or sequence similarity over the heavy chain variable region sequence to the heavy germline heavy chain variable sequence DP50 (which is an allele of the Vh 3-33 gene locus) or with the Vk A27 germline light chain variable sequence, or Vk 012 germline sequence. Even more preferably, the antibody heavy chain region sequence shares 100% sequence identity or sequence similarity with the germline sequence. DP50 or with the A27 germline light chain sequence or 012 germline sequence.

In one embodiment of the present invention, the antibody shares at least 80%, more preferably at least 85%, even more preferably at least 90%, still more preferably at least 95%, more preferably at least 99% of sequence identity or sequence similarity to heavy chain, light chain, or both, FR1 to FR4 sequences to antibody FR1 to FR4 region sequences 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13 .1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, 10D1. Even more preferably, the antibody shares 100% sequence identity or sequence similarity over heavy, light, or both sequences 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, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, and 10D1.

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

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%, even more preferably at least 95%, more preferably at least 99%, and preferably about 100% of sequence identity or sequence similarity with the FR1 through FR4 light chain sequences with the V1 A27 germline, or Vk 012 germline FR1 to FR4 region sequences.

In one embodiment of the present invention, the antibody shares at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, more preferably at least 99%. , sequence identity or sequence similarity to antibody heavy chain, light chain CDR-1, CDR-2 and CDR-3 sequences, or both, 3.1.1, 4.1.1, 4.8.1, 4.10. 2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, 10D1.

Even more preferably, the antibody shares 100% sequence identity or sequence similarity over the heavy, light, or both heavy 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, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, and10D1.

In another embodiment of the present invention, the antibody shares at least 80%, more preferably at least 85%, even more preferably at least 90%, even 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 DP50 germline CDR-1 and CDR-2 sequences.

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%, even more preferably at least 95%, more preferably at least 99%, and preferably about 100% sequence identity or sequence similarity to the light chain CDR-1, CDR-2 and CDR-3 sequences with the CDR-1, CDR-2, and CDR-3 sequences. germline Vk A27, or germline Vk 012.

Examples of antibodies employable in the present invention, and methods for producing them, are described, among others, in United States Patent Application No. 09 / 472,087, now issued as United States Patent No. 6,682,736; International Application No. PCT / US00 / 23356 (published March 1, 2001, as WO 01/14424) (e.g., antibody 10D1, also known as MDX-010, Medarex, Princeton, NJ); International Application No. PCT / US99 / 28739 (published June 8, 2000, as WO 00/32231); U.S. Patent No. 5,811,097, 5,855,887, 6,051,227, and 6,207,156; each of which is incorporated herein by reference. While information on nucleic acid and amino acid sequences referring to these antibodies is provided herein, other information can be found in United States Patent No. 6,682,736, as well as WO 00/37504; The sequences presented in these applications are hereby incorporated by reference.

Certain uses for these antibodies to treat various cancers have been described in United States Patent Application No. 10 / 153,382, now published as United States Patent Application Publication No. 2003/0086930, which is incorporated by reference as set forth. in its entirety here.

Features of human anti-CTLA4 antibodies useful in the methods of the invention are described extensively in, for example, United States Patent No. 6,682,736, and include antibodies that have 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, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, and 10D1. The invention also relates to methods employing antibodies comprising the heavy and light chain CDR amino acid sequences of these antibodies, as well as those comprising alterations in the CDR1 regions as described in the above cited applications and patents. The invention also relates to antibodies comprising the heavy and light chain variable regions of these antibodies. In another embodiment, the antibody is selected from an antibody comprising the full length, variable region, or CDR amino acid sequences of the antibody heavy and light chains 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13 .1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1, and 10D1.

While anti-CTLA4 antibodies described hereinbefore may be preferred, the skilled artisan, based on the description provided herein, would appreciate that the invention encompassed a wide variety of anti-CTLA4 antibodies and was not limited to these particular antibodies. . More particularly, while human antibodies are preferred, the invention is by no means limited to human antibodies; preferably, the invention encompasses useful antibodies, regardless of species origin, and includes, among others, chimeric, primatized and / or humanized antibodies. Furthermore, while the antibodies exemplified herein were obtained by employing a transgenic mammal, for example a mouse comprising a human immune repertoire, the skilled artisan, based on the description provided herein, would understand that the present invention is not limited to an antibody produced by this or any other particular method. In contrast, the invention includes an anti-CTLA4 antibody produced by any method, including, but not limited to, a method known in the art (e.g., evaluation of phage display libraries, and the like) or to be developed in the future. for the production of an anti-CTLA4 antibody of the invention. Based on the extensive description provided herein and, for example, United States Patent No. 6,682,736, by Hanson and the like, and United States Patent Application Publication No. 2002/0088014, one skilled in the art can readily produce and identifying an antibody useful for the treatment of breast cancer in combination with a hormonal therapeutic agent employing the novel methods described herein. The present invention encompasses human antibodies produced

employing a transgenic non-human mammal, i.e. XenoMouse® (Abgenix, Inc., Fremont, CA) as described in U.S. 6,682,736, by Hanson and the like.

Another transgenic mouse system for the production of "human" antibodies is referred to as the "HuMAb-Mouse®" (Medarex, Princeton, NJ), which contains human immunoglobulin gene miniscule encoding the kappa light chain immunoglobulin sequences. and undisposed human heavy (mu and gamma), together with target mutations that disable the kappa chain locus or an endogenous (Lonberg et al. Nature 368: 856-859 (1994), and US Patent No. 5,770,429).

However, the invention uses human anti-CTLA4 antibodies produced employing any transgenic mammal such as, but not limited to, Kirin TC Mouse® (Kirin Beer Kabushiki Kaisha, Tokyo, Japan) as described in, for example, Tomizuka and the like, Proe Natl Acad Sci USA 97: 722 (2000); Kuroiwa and the like, Nature Biotechnol 18: 1086 (2000); United States Patent Application Publication No. 2004/0120948, by Mikayama and the like; and HuMAb-Mouse® (Medarex, Princeton1 NJ) and Xe-noMouse® (Abgenix, Inc., Fremont, CA), supra. Accordingly, the a-brange invention will employ an anti-CTLA4 antibody produced employing any transgenic animal or other nonhuman animal.

In another embodiment, the antibodies employed in the methods of the invention are not fully human but "humanized". In particular, mouse antibodies or antibodies of other species may be "humanized" or "primatized" using techniques well known in the art. See, for example, Winter and Harris Immunol. Today 14: 43-46 (1993), Wright and the like Crit. Reviews on Immunol. 12: 125-168 (1992), and U.S. Patent No. 4,816,567, by Cabilly and the like, and Mage and Lamoyi in Monoclonal Antibody Produetion Teehniques and Applications. pp 79-97, Mareei 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, for example, US Patent Nos. 5,759,808 and 6,765,087, by Casterman and Hamers), as well as any human antibody, may be combined with a therapeutic agent to practice the novel methods described herein.

As will be appreciated from the description provided herein, antibodies for use in the invention may be obtained from a transgenic non-human mammal, and hybridomas derived therefrom, but may 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 available American Type Culture Collecton-on (ATCC) immortalized cell lines, including but not limited to Chinese hamster ovary cells. (CHO), NSO, Sp2, HEK, and HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). Eukaryotic and prokaryotic non-mammalian cells may also be employed, including bacterial, yeast, insect, and plant cells.

Various expression systems may be employed when well known in the art, such as, but not limited to those described in, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel and the like, Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002). These expression systems include dihydrofolate reductase (DHFR) based systems, among many others. The glutamine synthase expression system is described in whole or in part with respect to European Patent Nos. 0216846B1, No. 0256055B1, and No. 0323997B1, and European Patent Application No. EP89303964. In one embodiment, the antibody employed is made in NSO cells employing a glutamine synthetase (GS-NSO) system. In another embodiment, the antibody is made on CHO cells employing a DHFR system. Both systems are well known in the art and are described in, among others, Barnes and the like Biotech & Bioen-gineering 73: 261-270 (2001), and references cited herein.

Site-directed mutagenesis of the antibody CH2 domain to eliminate glycosylation may be preferred to prevent changes in any of the immunogenicity, pharmacokinetic, and / or effector functions resulting from non-human glycosylation. In addition, the antibody may be deglycosylated by enzymatic methods (see, for example, Thotakura and the like Meth. EnzymoL 138: 350 (1987)) and / or chemicals (see, for example, Hakimuddin and the like, Arco. Biochem. Biophys 259: 52 (1987)).

Further, the invention encompasses employing an anti-CTLA4 antibody comprising an altered glycosylation pattern. The skilled artisan would appreciate, based on the description provided herein, that an anti-CTLA4 antibody could be modified to comprise additional, smaller or different glycosylation sites compared to the naturally occurring antibody. Such modifications are described in, for example, United States Patent Application Publications Nos. 2003/0207336, and 2003/0157108, and International Patent Publication Nos. WO 01/81405 and 00/24893.

Additionally, the invention comprises employing a glycoform-independent anti-CTLA4 antibody, if any, present in the antibody. In addition, methods for extensively remodeling the glycoform present in a glycoprotein are well known in the art and include, for example, those described in International Patent Publication Nos. WO 03/031464, WO 98/58964, and WO 99/22764, and Patent Application Publications Nos. 2004/0063911, 2004/0132640, 2004/0142856, 2004/0072290, and United States Patent No. 6,602,684 by Umana and the like.

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

Additionally, the invention encompasses employing an anti-CTLA4 antibody, or antigen binding portion thereof, chimeric protein comprising, for example, a human serum albumin polypeptide, or fragment thereof. If the chimeric protein is produced by employing recombinant methods by, for example, cloning a chimeric nucleic acid encoding the chimeric protein, or by chemically linking the two peptide moieties, the skilled artisan would understand once armed with the teachings provided herein that such Chimeric proteins are well known in the art and may impart desirable biological properties such as, but not limited to, increased stability and serum half life for the antibody of the invention and such molecules will therefore be included herein.

Antibodies that are generated for use in the invention need not initially have a particular desired isotype. Preferably, the antibody when generated may have any isotype and may be switched isotype therefore employing conventional techniques. These include direct recombinant techniques (see, for example, United States Patent 4,816,397), and cell-cell fusion techniques (see, for example, United States Patent No. 5,916,771).

The effector function of the antibodies employed in the invention may be altered by switching the isotype to an IgGI, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeutic uses. In addition, complement dependence for cell death can be prevented by the use of bispecifics, immunotoxins, or radiolabels, for example.

Although antibody 4.1.1, 4.13.1 and 11.2.1 are IgG2 antibodies and antibody variable region sequences are provided herein (Figures 1-3), and in the applications and patents referenced and incorporated herein, it is understood that the Life-size 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 also comprising any isotype-independent constant regions as more fully described elsewhere herein. Likewise, any antibody comprising the 10D1 full-length sequence, or any portion thereof, including a sequence encoding a 10D1 antigen binding portion, may be employed 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 could comprise a broad plethora of anti-CTLA4 antibodies.

Furthermore, one skilled in the art, based on the description provided herein, would understand that the invention is not limited to the administration of only a single antibody; preferably the invention encompasses the administration of at least one anti-CTLA4 antibody, for example one of 4.1.1, 4.13.1, or 11.2.1, in combination with a therapeutic agent. Further, any combination of anti-CLTA4 antibodies may be combined with at least one therapeutic agent and the present invention encompasses any such combination and permutation thereof.

IV. Aromatase Inhibitor

The present invention relates to the administration of a combination of an anti-CTLA4 antibody and an aromatase inhibitor to a patient for breast cancer treatment.

Hormone therapy agents for treating estrogen receptor expression breast cancer tumors are well known in the art. These hormone therapy agents include compounds that affect the receptor itself, such as, but not limited to, tamoxifen which binds to the receptor thereby inhibiting the receptor-ligand interaction, and fulvestrant that degrades the receptor such that less is available. to mediate signaling by receptor interaction: ligand.

Additionally, hormone therapy agents comprise aromatase enzyme inhibitors such as, but not limited to, anas-trozol, letrozole, and exemestane. These antihormonal agents, unlike tamoxifen and fulvestrant, supra, inhibit receptor-binding interaction by decreasing the level of ligand available to bind with the receptor. More specifically, an aromatase inhibitor inhibits the A-ring aromatization of an androgen substrate to form an estrogen, which is a necessary step in receptor ligand biosynthesis, that is, estrogen. Thus, aromatase inhibitors measured decreased estrogen serum levels thereby inhibiting the growth of tumor cells that require growth hormone.

Non-steroidal aromatase inhibitors ("Al") such as, but not limited to, anastrozole and letrozole, reversibly bind to the heme portion of the aromatase molecule and are associated with an increase in aromatase activity. This may be associated with the development of tumor resistance, particularly with long term use of the inhibitor. In addition, estrogen biosynthesis in the presence of these inhibitors may occur without the need for new aromatase synthesis as long as the inhibitor is reversibly bound to the enzyme (see Miller Clin Breast Cancer 1: S9-S14 (2000)). also providing another opportunity for tumor cells to avoid inhibition.

Steroidal aromatase inhibitors, as for example, differ from non-steroidal inhibitors (eg, anastrozole and letrozole) in that steroidal agents bind irreversibly to the enzyme substrate binding site and are associated with a decrease in aromatase activity. For example, exemestane is structurally related to natural substrate androstenedione, and is recognized by the enzyme as a false substrate such that it competes with the natural substrate at the active site. Exemestane is then transformed into an intermediate that irreversibly binds to the enzyme causing its deactivation (ie, "suicide inhibition"). See, for example, United States Patent Application No. 10 / 611,653, published as United States Patent Application Publication No. 2004/0082557, United States Patent Application No. 10 / 363,935, published as Application Publication United States Patent Application No. 2004/0024044, and United States Patent Application No. 10 / 343,595, published as United States Patent Application Publication No. 2003/0158168.

Thus, unlike reversible non-steroidal inhibitors, exemestane is a steroidal aromatase deactivator that irreversibly binds aromatase, causing almost complete suppression of aromatase activity in peripheral as well as tumor, and mediates substantial reduction. of serum estrogen levels without extraneous endocrinological effects (Kaufmann et al. J Clin Oncol 18: 1399-1411 (2000)). Exemestane is currently approved in the US, Canada, and Europe for second-line treatment (eg, where the disease has progressed following tamoxifen first-line therapy) in postmenopausal women with metastatic breast cancer. Exemestane produces a response rate of approximately 15% with an average survival of 123.4 weeks and demonstrates a lack of inter-resistance with non-steroidal agents such that it has been shown to be effective following treatment with anastrozole and etrozole (Geisler et al. Clin. Cancer Res 4: 2089-2093 (1998); Lonning J Clin Oncol 18: 2234-2244 (2000)). More recently, exemestane has been approved in Europe for adjuvant treatment of estrogen receptor positive invasive early breast cancer following two to three years of initial adjuvant tamoxifen therapy in postmenopausal women.

Because its intermediate is irreversibly linked, exemestane, unlike non-steroidal aromatase inhibitors, also prevents estrogen biosynthesis by the fact that de novo synthesis of aromatase enzyme is required. Furthermore, the different mechanism of action of non-steroidal and steroidal aromatase inhibitors may explain the phenomenon that one aromatase inhibitor may be effective following the failure of the other. More particularly, the treatment of postmenopausal breast cancer patients treated with exemestate following the failure of both tamoxifen and a non-steroidal aromatase inhibitor provided a clinical benefit (Lonning et al. J Clin Oncol 18: 2234-2244 (2000)). ).

Thus, anti-CTLA4 antibody in combination with any aromatase inhibitor, or multiple inhibitors, may be employed to treat a breast cancer patient where the tumor expresses estrogen receptor (ER +). Furthermore, while the present invention is exemplified by a combination comprising exemestane, the invention is by no means limited to this inhibitor or mechanism of inhibition thereof. Indeed, the mechanism by which the aromatase inhibitor inhibits the enzyme need not be known. Thus, any inhibitor that noticeably inhibits the ring A aromatization of a substrate in estrogen biosynthesis employing any assay known to evaluate such activity may be employed in the combination of the invention. V. Additional Agents

The present invention may also be combined with additional agents and therapies, for example chemotherapy, surgery, radiotherapy, transplantation, and the like, to treat a patient. That is, the patient may undergo additional chemotherapy with well-known agents, such as, but not limited to, growth factor inhibitors, biological response modifiers, alkylating agents, intercalation antibiotics, vinca. alkaloids, immunomodulators, taxanes, selective estrogen receptor modulators (SERMs) 1 such as, but not limited to, Iasofoxife-1, and angiogenesis inhibitors.

Therapeutic agents are numerous and have been described, for example, in United States Patent Publication No. 2004/0005318, No. 2003/0086930, No. 2002/0086014, and International Publication No. WO 03/086459, all of which are incorporated herein by reference herein, among many others. Such therapeutic agents include, but are not limited to, topoisomerase I inhibitors; other antibodies (rituxi-mab, 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 (GLEE-VEC), SU11248 (SUTENT; sunitinib), SU12662, SU14813; BAY 43-9006, AG-013736 (axitinib), toll-like receptor stimulating agents (eg, TLR-9 agonist; such as, but not limited to, CPG-7909, also referred to as PF03512676 or PROMUNE), indolein-2,3-dioxoxygenase (IDO) inhibitors; selective estrogen receptor modulators (SERMs; for example, lasofoxifene); taxanes; vinca alkaloids; temozoolomide; angiogenesis inhibitors; EGFR inhibitors; VEGF inhibitors; erbB2 receptor inhibitors; antiproliferative agents (e.g., farnesyl protein transferase inhibitors, and ανβ3 inhibitors, ανβ5 inhibitors, p53 inhibitors, and the like); immunomodulators; biological response modifiers; cytokines; tumor vaccines; specific tumor antigens; heat shock protein-based tumor vaccines; stem cell and dendritic 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, 0x40, ICOS1 CD30, HLA-DR, MHCII1 and LFA), and agonist antibodies as well; among many others.

In one embodiment, the methods of the invention may also be combined with transplantation, for example stem cell transplantation, to provide a therapeutic benefit to a patient afflicted with breast cancer. Stem cell transplantation may be performed according to methods known in the art and may be allogeneic or autologous stem cell transplantation. Additionally, one of ordinary skill in the art would appreciate, based on the description provided herein, that the transplantation encompasses adoptive transfer of lymphocytes, either autologous or obtained from a combined HLA donor. Where the method comprises stem cell transplantation, the first dose of the AI-antibody therapy agent combination may be administered after the mammalian immune system has recovered from transplantation, for example, within one to 12 months post-transplantation. -transplantation. In certain embodiments, the first dose is administered within one to three, or one to four months after transplantation. Transplantation methods describe many treatises, including Appelbaum in Harrison's Principles of International Medicine, Chapter 14, Braunwald and the like, Eds., 15th ed., McGraw-HiII Professional (2001), which is hereby incorporated by reference.

As previously noted herein, there are many chemotherapeutic agents currently available for treating 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 the unwinding and replication of strains. The alkylating agent for use in the methods of the present invention is cyclophosphamide (CYTOXAN).

Folate antagonists bind to dihydrofolate reductase (DHFR) and interfere with the synthesis of pyrimidine (thymidine). Methotrexate and peme-trexed (ALIMTA) are folate antagonists suitable for use in the methods of the present invention. In addition to DHFR, pemetrexed also inhibits glycinamide transferase ribonucleotide thymidylate synthase and formyl, two other folate-dependent enzymes involved in thymidine synthesis.

Pyrimidine antagonists inhibit the 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. Suitable pyrimidine antagonists for use in the methods of the present invention include 5-fluorouracil (5-FU); capecitabine (XELODA), a 5'-deoxy-5-fluorouridine (5-FDUR) prodrug that is enzymatically converted to 5-FU in vivo; and gemcitabine (GEMZAR).

Anthracycline antibiotics inhibit DNA unwinding by interleaving between DNA strains. 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 antineoplastic effect by intercalating DNA strains, which inhibits DNA unwinding. Platinum compounds useful in the methods of the present invention include cisplatin (PLATINOL), oxaliplatin (ELOXATIN), and carboplatin (PARAPLA-TIN).

Taxanes promote microtubule assembly while inhibiting their disassembly into tubulin, thereby blocking a cell's ability to decompose the mitotic spindle during mythology. They have shown 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-1R antibody. Suitable taxanes for use in combination with the IGF-1R antibody include docetaxel (TAXOTERE) and paclitaxel (TAXOL).

Taxane derivatives that can be active on doxorubicin, vinblastine, paclitaxel, docetaxel, and the like resistant cells include XRP-9981 (Sanofi Aventis), and are encompassed by the invention.

Vinca alkaloids, like taxanes, are "spindle poisons," acting on the microtubules that make up the mitotic spindle. They inhibit mitosis by interfering with the microtubule assembly, preventing the spindle from forming. Vinca alkaloids include vindesine (ELDISINE), vinblastine sulfate (VELBAN), vincristine sulfate (ONCOVIN) and vinorelbine tartrate (NA-VELBINE). A preferred alkaloid vinca 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.

Rapamycin analogs, which bind and inhibit the rapamycin target mammal (mTOR), are also useful and include, but are not limited to, CCI-779 (temsirolimus; Wyeth) and RAD-001 (everolimus, CERTICAN; Novartis).

Camptothecin analogs act by inhibiting topoi somerase I, a critical enzyme for DNA replication and packaging. Topoisomerase I levels are higher in tumor cells than in normal tissue. A camptothecin analog useful in the methods of the present invention is irinotecan (CAMPTOSAR).

In certain embodiments of the invention, the methods described above are combined with a cancer vaccine. See, for example, Oh and the like, Cancer Res. 64: 2610-2618 (2004) (TARP epitopes and breast cancer); Kontani et al., Int J Molec Med 12: 493-502 (2003) (MUC1 mucin targeting dendritic cell vaccine); Holmberg & Sandmaier Ex-pert Rev Vaccines 3: 269-277 (2004) (breast or ovarian cancer vaccination). That is, useful vaccines may be, without limitation, those comprised of breast cancer-associated antigens (eg, HER-2 / neu, gammaglobin, prostate and breast tumor-associated protein [TARP], MUC1, CEA, sialil -Tn and similar carbohydrate antigens), other tumor cancer-associated antigens (eg, p53, telomerase), anti-biotype antibodies such as 11D10, as well as vaccines comprising GM-CSF, DNA and cell-based vaccines, vaccines of dendritic cells, recombinant viral vaccines (e.g., vaccinia virus), and heat shock protein (HSP) vaccines (e.g., HSPPC-96; Antigenics Inc.). Useful vaccines also include tumor vaccines, such as those formed from breast tumor cells; and may be autologous or allogeneic, and vaccines may be cell, peptide, or DNA based.

Vaccines may be administered prior to or subsequent to administration of the antibody-aromatase inhibitor combination, and when chemotherapy is part of the regimen, a vaccine may be administered prior to chemotherapy. In certain embodiments, the antibody-aromatase inhibitor combination of the invention may also be administered prior to chemotherapy. In still other embodiments, the treatment may be combined with stem cell transplantation. That is, the antibody-aromatase inhibitor combination may be administered before or after stem cell transplantation. The vaccine may also be administered before or after stem cell transplantation and, in certain embodiments, concomitantly with the antibody.

The treatments described above may also be employed with signal transduction inhibitors, such as agents that may inhibit EGFR (epidermal growth factor receptor) responses, such as EGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors. ; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF receptors and molecules that can inhibit VEGF; and erbB2 receptor (HER2) inhibitors such as organic molecules or antibodies that bind to the erbB2 receptor, for example trastuzumab (HERCEPTIN, Genentech, Inc., San Francisco, CA), and pertuzumab (2C4, OMNITARG; Ge -nentech) which is a HER dimerization inhibitor (HDI).

EGFR inhibitors are described, for example, in International Patent Publication Nos. WO 95/19970, WO 98/14451, WO 98/02434, and United States Patent No. 5,747,498, and such substances may be employed in the present invention as described herein. EGFR inhibiting agents include, but are not limited to, C225, anti-EGFR 22Mab monoclonal antibodies (ImCIone System Inc., New York, NY), and ABX-EGF (Abgenix Inc., remont, CA), the compounds ZD-1839 (Astra-Zeneca), BIBX-1382 (Boehringer Ingelheim), MDX-447 (Medarex1Inc., Annandale, NJ), and OLX-103 (Merck & Co., Whitehouse Station1 NJ), VRCTC-310 ( Ventech Research) and EGF fusion toxin (Seragen Inc., Hopkinton1 MA). These and similar EGFR inhibiting agents may be employed in the present invention.

Compounds targeting epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibition 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 triggers a cascade of cellular reactions that result in increased cell division and influences other aspects of cancer development and progression, including angiogenesis, metastatic expansion, and inhibition of apoptosis. EGFR-TK inhibitors can selectively target one of the EGFR family members (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 you can target two or more of them. Suitable EGFR-TK inhibitors for use in the present invention include gefitinib (IRESSA), erlotinib (TARCEVA), CI-1033 (Pfizer), GW2016 (GIaxoSmithKIine), EKB-569 (Wyeth), PKI-166 (Novartis), CP -724,714 (Pfizer), and BIBX-1382 (Boeringer-Ingelheim). Additional EGFR-TK inhibitors are described in United States Patent No. 6,890,924.

VEGF inhibitors, for example SU-5416, SU-6668, SU-11248, SU-12662, SU-14813 (Sugen Inc., San Francisco, CA) as well as AG-013736 (Pfizer) may also be employed. in combination with the antibody. VEGF inhibitors are described, for example, in International Patent Application No. PCT / IB99 / 00797 (filed May 3, 1999), International Patent Publication Nos. WO 99/24440; WO 95/21613; WO 99/61422; WO 98/50356; WO 99/10349; WO 97/32856; WO 97/22596; WO 98/54093; WO 98/02438; WO 99/16755; WO 98/02437; United States Patent Nos. 5,834,504; 5,883,113; 5,886,020; and 5,792,783. Other examples of some specific VEGF inhibitors useful in the present invention are IM862 (Cytran Inc., Kirkland1 WA); IMC-1C11 antibody Imclone, anti-VEGF monoclonal antibody from Genentech, Inc., San Francisco, CA; and angiozyme, a synthetic ribozyme from Ribozyme (Boul-der, CO) and Chiron (Emeryville, CA).

ErbB2 receptor inhibitors such as GW-282974, GW-572016 (Iapatinib) (Wellcome Glaxo pie), and AR-209 monoclonal antibodies (Aronex Pharmaceuticals Inc., Woodlands, TX), trastuzumab (HER-CEPTIN; Genentech , Inc., San Francisco, CA), pertuzumab (OMNITARG; 2C4; Genentech, a HER2 HDI dimerization inhibitor), and 2B-1 (Chiron) 1 may be combined with antibody-aromatase inhibitor combination therapy. Other erbB2 receptor inhibitors are described, for example, in International Patent Publication Nos. WO 98/02434; WO 99/35146; WO 99/35132; WO 98/02437; WO 97/13760; WO 95/19970; 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 International Patent Publication No. WO 00/44728 (published August 3, 2000). The erbB2 receptor inhibitor compounds and substance described in the above mentioned PCT applications, US Patent, and United States provisional applications, as well as other erbB2 receptor inhibiting compounds and substances, may be employed with the combination of inhibitor of aromatase and antibody according to the present invention.

Treatments of the invention are also employed with other agents useful in the treatment of cancer or abnormal cell growth, including, but not limited to, other agents capable of enhancing antitumor immune responses, such as additional, different CTLA4 antibodies, and the like. also capable of blocking CTLA4; and antiproliferative agents such as farnesyl protein transferase inhibitors, and ανβ3 inhibitors, such as ανβ3 antibody VITAXIN, ανβ5 inhibitors, p53 inhibitors, and the like.

Where the antibody of the invention is administered in combination with another immunomodulatory agent, the immunomodulatory agent may be selected from, for example, the group consisting of a dendritic cell activator such as CD40 ligand and anti-CD40 agonist antibodies, as well as antigen presentation enhancers, T cell tropism enhancers, inhibitors of tumor-related immunosuppressive factors such as TGF-β (transforming growth factor beta), and IL-10. Preferred anti-CD40 agonist antibodies encompass the antibodies described in International Patent Application No. PCT / US02 / 36107, filed November 8, 2002, now published as International Patent Publication No. WO 03/040170, and Patent Application. No. 10 / 292,088, filed November 8, 2002, now published as United States Patent Publication No. US2003 / 0211100, including, but not limited to, an antibody that has the chain amino acid sequence heavy and light weight of 3.1.1, 3.1.1.H-A78T, 3.1.1H-A78T-V88A-V97A, 3.1.1L-L4M-L83V, 3.1.1 H-A78T-V88A-V97A / 3.1.1L- L4M-L83V, 7.1.2, 10.8.3, 15.1.1, 21.2.1, 21.4.1, 22.1.1, 22.1.1H-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-1R (insulin-like growth factor 1 receptor). Specific anti-IGF-1R antibodies that may be employed 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. W002 / 053596, International Patent Application No. PCT / IB2004 / 002555, filed August 3, 2004, and published as International Patent Publication No. WO 2005/016967. Preferred anti-ti-IGFR-1R antibodies comprise an antibody having the heavy and light chain amino acid sequence of, for example, antibody 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3.

The antibody of the invention may also be administered with cytokines such as IL-2, interferon (e.g., IFN-γ, IFN-α, etc.), GM-CSF, IL-12, IL-18, and FLT- 3L.

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 may be employed together with the antibody in the method of the invention. Examples of useful COX-II inhibitors include CELEBREX (celecoxib), valdecoxib, and rofecoxib. 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 Applications Nos. 780386 (published June 25, 1997), 97304971 (filed July 8, 1997), 99308617 (filed October 29, 1999), 606046 (published July 13, 1994), 931788 (published 28 July 1997). July 1999), 99302232 (filed March 25, 1999), International Application PCT / IB98 / 01113 (filed July 21, 1998), United Kingdom patent application number 9912961 (filed June 3, 1999 ), United States Provisional Patent Application No. 60 / 148,464 (filed August 12, 1999), and US Patent Nos. 5,863,949, and 5,861,510.

Preferred MMP-2 and MMP-9 inhibitors are those that have little or no MMP-1 inhibiting activity. Most preferred are those that selectively inhibit MMP-2 and / or MMP-9 relative to other matrix meta-Ioproteinases (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, and the combinations 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-hydroxycarbo-moyl-cyclobutyl) -amino] -propionic acid;

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

(R) 3- [4- (4-Chloro-phenoxy) -benzenesulfonamino] -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-fluoro-phenoxy) -benzenesulfonyl] - (1-hydroxycarbo-moyl-1-methyl-ethyl) -amino] -propionic acid;

3 - [[4- (4-fluoro-phenoxy) -benzenesulfonyl] - (4-hydroxycarbo-moyl-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;

(R) 3- [4- (4-Fluoro-phenoxy) -benzenesulfonylamino] -tetrahydro-furan-3-carboxylic acid hydroxyamide;

and pharmaceutically acceptable salts and solvates of said compounds.

Radiation therapy may be administered according to well-known radiotherapy methods for treating breast cancer. The dose and regimen for radiotherapy can easily be determined by one skilled in the art and is based on the stage of the disease, and similar factors well known in the art.

Coadministration of the antibody with an aromatase inhibitor (combination therapy) comprises administration of a pharmaceutical composition also comprising the anti-CTLA4 antibody and one or more aromatase inhibitor (s), and administration of two or more separate pharmaceutical compositions. one comprising the anti-CTLA4 antibody and the other comprising the aromatase inhibitor (s). In addition, while coadministration or combination therapy generally means that the antibody and additional therapeutic agents are administered at the same time as one another, it also encompasses simultaneous, separate or sequential dosages of individual treatment components. Additionally, where an antibody is administered intravenously and the aromatase inhibitor 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 similar

therapeutic agents beyond the first and second component, or concurrently with one or more of these components, or sequentially before and / or after. Such therapeutic agents include cancer vaccines, anti-vascular agents, antiproliferative agents, and palliative agents to provide supportive care such as, but not limited to, analgesics, antiemetic agents, antidiarrheal agents, and steroids. Preferred antiemetic agents include ondansetron hydrochloride, granise-tron hydrochloride, and metoclopramide. Preferred antidiarrheal agents include diphenoxylate and atropine (LOMOTIL), Ioperamide (IMMODIUM), octreotide (SANDOSATATIN), olsalazine (DIPENTUM), and mesalamine (ASACOL). Preferred steroids include non-absorbable steroid budesonide (ENTOCORT), and systemic administration dexamethasone (DECADRAN) and prednisone (METI-CORTEN) steroids.

Each administration may vary in duration from rapid administration to continuous infusion. As a result, for the purpose of the present invention, combinations are not exclusively limited to those which are obtained by physical association of the constituents, but also to those which permit separate administration, which may be simultaneous or spaced over a period of time. The compositions according to the invention are preferably parenterally administered compositions. However, these compositions may be administered orally or intraperitoneally for localized regional therapies.

As will be appreciated by one of ordinary skill in the art, the choice of therapeutic agents to be employed in combination with aromatase inhibitor and anti-CTLA4 antibody combination therapy, and the duration of their use, will be determined in part by type and stage. of the cancer being treated. For example, in early breast cancer (where cancer has not spread outside the breast), surgery and radiation are usually followed by adjuvant chemotherapy or adjuvant hormone therapy, any of which may be combined with the aromatase-inhibitor combination. anti-CTLA4 antibody in the methods of the present invention. Typical adjuvant chemotherapy for early breast cancer includes cyclophosphamide, methotrexate and 5-FU ("CMF"); 5-FU, doxorubicin, and cyclophosphamide ("FAC"); docetaxel, doxorubicin, and cyclophosphamide ("TAC"); doxorubicin and cyclophosphamide ("AC"); doxorubicin and cyclophosphamide followed by paclitaxel ("AC and T"); and 5-FU, epirubicin, and cyclophosphamide ("FEC"). As previously described elsewhere herein, tamoxifen is an antihormonal treatment administered at this stage. Thus, the antibody-aromatase inhibitor combination may be administered in combination with additional hormone therapy (e.g., another aromatase inhibitor, tamoxifen, fulvestrant, or any combination thereof), and such combination may be co-administered in combination. adjuvant chemotherapy therapy (e.g., CMF, FAC, TAC, AC1 AC and T, and / or FEC, or chemotherapeutic agents may be administered individually in combination with the antibody-aromatase inhibitor combination, among others). ).

In locally advanced breast cancer, where cancer has spread only to nearby lymph nodes or tissues, the patient is often given chemotherapy before surgery and radiation, which is then followed by adjuvant hormone therapy. Alternatively, surgery / radiation is followed by adjuvant chemotherapy, then adjuvant hormone therapy. The combination of anti-CTLA4-aromatase inhibitor antibody may be administered in conjunction with chemotherapeutic or additional hormone therapy agents (for example, another aromatase inhibitor, tamoxifen, fulvestrant, or any combination thereof) if they are used. employed or before or after surgery / radiation. Typical chemotherapy regimens for locally advanced breast cancer include FAC, AC, ECF1 and doxorubicin plus docetaxel ("TA").

Metastatic breast cancer has spread to other parts of the body from the breast in which it began. Chemotherapy may optionally be preceded by hormone therapy. First-line hormone therapy currently includes tamoxifen and anastrozole. First-line chemotherapy regimens include FAC, TAC, docetaxel plus epirubicin, docetaxel, paclitaxel, capecitabine, vinorelbine, and trastuzumab (HERCEP-TIN). Second-line treatments include docetaxel alone or in combination with capecitabine. The methods of the present invention are suitable for use both as first line therapy and second line therapy. In addition, the methods of the invention may be combined with radiation therapy and stem cell transplantation, and any combination of any of the treatments described herein, known in the art, or to be developed in the future.

In one embodiment, the additional therapeutic agent administered with aromatase inhibitor antibody therapy is an alkylating agent. In one aspect, the alkylating agent is cyclophosphamide.

In another embodiment, the additional agent is a leaflet antagonist. In one aspect, the folate antagonist is selected from the group consisting of methotrexate and pemetrexed.

In one embodiment of the invention, the additional agent is a pyrimidine antagonist. The pyrimidine analog may be selected from the group consisting of 5-FU, capecitabine, and gemcitabine.

In another embodiment of the present invention, the additional agent is an anthracycline antibiotic. In one aspect, the anthracycline antibiotic is selected from the group consisting of epirubicin and doxorubicin. In another aspect, the anthracillin antibiotic is doxorubicin.

In another embodiment of the invention, the additional agent is a platinum compound. The platinum compound can be selected from the group consisting of cisplatin and carboplatin.

In yet another embodiment of the invention, the additional agent is a taxane. In one aspect, the taxane is docetaxel. In another aspect, the rate is not paclitaxel.

In one embodiment of the present invention, the combination of anti-CTLA4 antibody and aromatase inhibitor is administered with docetaxel in additional combination with at least one of capecitabine, cisplatin, gemcitabine, and epirubicin. In one embodiment of the invention, the combination of anti-CTLA4

and aromatase inhibitor is administered in additional combination with paclitaxel and an additional agent selected from the group consisting of carboplatin, cisplatin, and gemcitabine. In one aspect, the additional agent is carboplatin.

In another embodiment, the combination of anti-CTLA4 and aromatase inhibitor is administered with an additional agent wherein the additional agent is an alkaloid vinca. In one aspect, the alkaloid vinca is vinorelbine.

In one embodiment of the invention, the additional agent administered with the antibody-aromatase inhibitor combination is a camptothecin analog. In one aspect, the camptothecin analog is irinotecan.

In another embodiment, the additional agent is an EGFR inhibitor.

In one embodiment of the invention, the additional agent is an erbB2 inhibitor. In one aspect, the erbB2 inhibitor is selected from trastu-zumab and pertuzumab. In yet another aspect, the erbB2 inhibitor is trastuzumab (HERCEPTIN).

In one embodiment of the invention, the combination of anti-CTLA4 and aromatase inhibitor is administered in further combination with a combination comprising 5-FU, doxorubicin, and cyclophosphamide.

In another embodiment of the present invention, the combination of anti-CTLA4 and aromatase inhibitor is administered in further combination with a combination comprising docetaxel, doxorubicin, and cyclophosphamide. The methods of the present invention also relate to treating cancer in a mammal, preferably a human, who has undergone stem cell transplantation, which methods comprise administering to the mammal an amount of a human antiCTLA4 antibody that is effective in treating of cancer in combination with an aromatase inhibitor in additional combination with stem cell transplantation. Cell transplantation may be allogeneic or autologous stem cell transplantation, and more preferably, the aromatase inhibitor is exemestane. Where the method comprises stem cell transplantation, the first dose of the antibody-aromatase inhibitor combination may be administered after the mammalian immune system has recovered from the transplantation, for example, within one to 12 months post transplantation. In certain embodiments, the first dose is administered within one to three, or one to four months after transplantation. The patient may undergo preparatory treatment (s) for stem cell transplantation.

SAW. 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 an aromatase inhibitor, preferably a steroidal aromatase inhibitor, and even more preferably, the aromatase inhibitor is exemestane. . Such a pharmaceutical composition may consist of each active ingredient only, as a combination of at least one active ingredient (e.g., an effective dose of an anti-CTLA4, an effective dose of an aromatase inhibitor) in a form suitable for administration to an individual, or pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients (active and / or inactive), or some combinations thereof.

In one embodiment, the antibody is administered parenterally (e.g. intravenously) in an aqueous solution while the aromatase inhibitor (e.g. exemestane) is administered orally in pill / capsule form. However, the skilled artisan would understand from the description provided herein that the invention is not limited to these or any other formulations, doses, administration routines, and the like. Preferably, the invention encompasses any formulation or method of administering an antibody in combination with an aromatase inhibitor, including, but not limited to, administering each agent separately in a different formulation by a different administration routine, and administering the antibody. and aromatase inhibitor in a single composition (e.g., in an administered aqueous composition, inter alia, iv), among many others. Accordingly, the following description describes various formulations for practicing the methods of the invention comprising administering any anti-CTLA4 antibody in combination with any aromatase inhibitor, but the invention is not limited to these formulations, but comprises any formulation as may be readily apparent. determined by one skilled in the art as equipped with the teachings provided herein for use in the methods of the invention.

Antibodies employed in the invention may be incorporated into pharmaceutical compositions suitable for administration to an individual. 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, absorption delaying and isotonic agents, and the like which 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 minor or wetting amounts of auxiliary substances such as emulsifying or wetting agents, preservatives or buffers, which enhance the shelf life or efficacy of the antibody or antibody moiety.

Antibodies can be in a variety of forms. These include, for example, solid, semi-solid liquid 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 intended mode of administration and therapeutic application. Typical preferred compositions are in the form of infusible or injectable solutions, such as compositions similar to those employed 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 infusion or intravenous injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high drug concentration. Sterile injectable solutions may be prepared by incorporating antibody in the required amount into an appropriate solvent with one or a combination of ingredients listed above as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients of those enumerated above. In the case of sterile powder for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization which produces an active ingredient powder plus any additional desired ingredient of a previously sterile filtered solution. The proper flowability of a solution may be maintained, for example, by the use of a coating such as lecithin, by maintaining the particle size required in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions may be accomplished by including in the composition an agent that delays absorption, for example monostearate and gelatin salts.

Antibodies may be administered by a variety of methods known in the art, including, without limitation, oral, parenteral, mucosal, inhalation, topical, buccal, nasal, and rectal. For many therapeutic applications, the routine / preferred mode of administration is subcutaneous, intramuscular, intravenous or infusion. Needle-free injection may be employed if desired. As will be appreciated by the skilled artisan, the mode and / or routine 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 from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated release systems. Biodegradable, biocompatible polymers may be employed, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing 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., Mareei Dekker, Inc., New York (1978). Dosage regimens may be adjusted to provide the best

ideal desired answer. For example, a single bolus may be administered, multiple divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the demands of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form to facilitate administration and uniformity of dosage. Dosage unit form as employed herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for unit dosage forms of the invention is dictated by and directly dependent upon (a) the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be obtained, and (b) the limitations inherent in the art of compounding technique. Such an active compound for the treatment of sensitivity in individuals.

It will be appreciated 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 also to be understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges presented herein are exemplary only and is not intended to limit the scope or practice of the claimed composition.

In one embodiment, the antibody is administered in an intravenous formulation as a sterile aqueous solution containing 5 mg / ml, or more preferably about 10 mg / ml, or even more preferably about 15 mg / ml, or even more preferably. , about 20 mg / ml 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. In addition, a solution comprising an anti-CTLA4 antibody may 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.

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

With respect to an aromatase inhibitor, the inhibitor may be present in the 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 further developed in the art of pharmacology. In general, such preparative methods include the step of bringing the active ingredient in 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 multiple or single dose unit.

Pharmaceutical compositions which 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 other routine administration. Other contemplated formulations include projected nanoparticles, liposome preparations, resealed erythrocytes containing the active ingredient, and immunologically based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold by volume, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a 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 active ingredient that would be administered to an individual or a convenient fraction of such a dosage, 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 ingredient in a pharmaceutical composition of the invention will vary depending upon the identity, size, and condition of the subject treated and also upon the routine 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 also comprise one or more additional pharmaceutically active agents. Additional agents particularly contemplated include antiemetic, antidiarrheal, chemotherapeutic, cytokine, and the like.

Controlled or extended release formulations of a pharmaceutical composition of the invention may be made employing conventional technology.

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

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

A tablet comprising the active ingredient may, for example, be made by pressing or molding the active ingredient, optionally with one or more additional ingredients. Pressed tablets may be prepared by pressing, 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 surfactant, 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 employed in tablet manufacture 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 surfactants include, but are not limited to, sodium lauryl sulfate. 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, cornstarch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pregelatinized cornstarch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

The tablets may be uncoated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of an individual thereby providing prolonged release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be employed to coat the tablets. Also by way of example, tablets may be coated using methods described in U.S. Patent Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. The tablets may also comprise a sweetening agent, flavoring agent, coloring agent, a preservative, or some combinations thereof to provide pharmaceutically elegant and flavorful preparation.

Hard capsules comprising the active ingredient may be made employing a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may also 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 employing 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 by employing conventional methods for suspending the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, isotonic saline and water. Oily carriers include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may also comprise one or more additional ingredients including, but not limited to suspending agents, wetting or dispersing agents, emulsifying agents, demulcentants, preservatives, buffer, salts, flavors, coloring agents, and sweetening agents. Oily suspensions may also comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl. Known wetting or dispersing agents include, but are not limited to, naturally occurring phosphatides such as lecithin, fatty acid condensation products of an alkylene oxide, a long chain aliphatic alcohol, a partial ester derived from a fatty acid and a hexitol, or a partial ester derived from a fatty acid and a hexitol amide (e.g., polyoxyethylene stearate, heptadecaethylene oxyethanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbi tan monooleate, respectively). Known emulsifying agents include, but are not limited to, Iecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl parahydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, saccharose, and saccharin. Known thickening agents for oil 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 main 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 respect to liquid suspensions, it being understood that suspending agents will not necessarily assist in dissolving the active ingredient in the solvent. Aqueous solvents include, for example, isotonic saline and water. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powder and granular formulations of a pharmaceutical preparation of the invention may be prepared employing known methods. Such formulations may be administered directly to an individual, employed, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by adding an aqueous or oily carrier thereto. Each of these formulations may also comprise one or more wetting and dispersing agents, suspending agents, and a preservative. Additional excipients, such as fillers and wetting, 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 a water in oil emulsion or an oil in water emulsion. The oil phase may be a vegetable oil such as olive or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions may also comprise one or more emulsifying agents such as naturally occurring gums such as acacia or tragacanth gum, naturally occurring phosphatides such as Iecithin or soybean phosphatides, esters or partial esters derived from fatty acid and anhydride combinations. hexitol such as sorbitan monooleate, and condensation products of such ethylene oxide partial esters 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 colonic or rectal 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 20 ° C) 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 also comprise a number of 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 employing, and may be packaged within, a delivery device adapted to the individual's rectal anatomy. Enema preparations may also comprise a number of 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, a coated or impregnated vaginally insertable material such as a tampon, a shower preparation, or gel or cream or a solution for vaginal irrigation.

Methods for impregnating or coating a material with

A chemical composition is known in the art, and includes, but is not limited to methods of depositing or attaching a chemical composition to a surface, methods of incorporating a chemical composition into the structure of a material during material synthesis (i.e. as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension in an absorbent material, with or without subsequent drying.

Shower preparations or vaginal irrigation solutions may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, shower preparations may be administered employing, and may be packaged within, a delivery device adapted to the subject's vaginal anatomy. Shower preparations may also comprise a number of additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.

As used herein, "parenteral administration" of a pharmaceutical composition includes any administration routine characterized by physical opening of a tissue of an individual and administration of the pharmaceutical composition through opening in the tissue. Parenteral administration thus includes, but is not limited to, administering a pharmaceutical composition by injecting the composition, applying the composition through a surgical incision, applying the composition through a non-surgical tissue penetrating wound, and the like. . In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and dialysis infusion of the kidney techniques.

Formulations of a suitable pharmaceutical composition 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 manner suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in a multi-dose container containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable biodegradable or extended release formulations as described below. Such formulations may also 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 form (i.e., powder or granular) 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 may be administered by a variety of methods known in the art. The mode and / or routine of administration varies depending on the desired results. Active compounds may be prepared with vehicles that protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated release systems. Biodegradable, biocompatible polymers may be employed, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are described by, for example, Sustained and Controlled Release Drug Delivery Systems, J., R. Robinson, ed., Mareei Dekker, Inc., New York, (1978). The 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 by employing a parenterally acceptable non-toxic diluent or solvent, such as water or diol 1,3-butane, 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 diglycerides. Other useful parenterally formulable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable hydrophobic or polymeric materials such as an emulsion, an ion exchange resin, a rarely soluble polymer, or a rarely 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% (weight / weight) of the active ingredient, although the active ingredient concentration may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may also 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 cavity administration. Such a formulation may comprise dry particles comprising the active ingredient and having a diameter in the range of 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 powder for administration employing a device comprising a dry powder reservoir so that a propellant stream may be directed to disperse the powder or employing a self-propelled powder / solvent dispersion container. such as a device comprising the active ingredient dissolved or suspended in a low boiling propellant in a sealed container. Preferably, such a powder comprises 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 of 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 of less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in unit dose form.

Low boiling impellers generally include liquid propellants having a boiling point below 18.33 ° C (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 also comprise additional ingredients such as a solid nonionic or anionic liquid 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 release 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 optionally sterile aqueous or dilute aqueous solutions or suspensions comprising the active ingredient, and may conveniently be administered by employing any nebulizing or atomizing device. Such formulations may also comprise one or more additional ingredients including, but not limited to, the flavoring agent such as saccharin sodium, a volatile oil, a buffered agent, a surfactant, or a preservative such as methylhydroxybenzoate. The droplets provided by this administration routine preferably have an average diameter in the range of from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary release are also useful for intranasal release 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 of about 0.2 to 500 micrometers. Such a formulation is administered in such a manner that inhalation is taken, that is, by rapid inhalation through the nasal passage of a powder container held close to the nostrils.

Formulations suitable for nasal administration may, for example, comprise from as little as 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may also 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) of the active ingredient, equilibrium comprising an orally composition. dissolvable or degradable and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder or an atomized or aerosolized suspension or solution comprising the active ingredient. Such aerosolized or aerosolized powder formulations when dispersed preferably have an average particle or droplet size in the range of about 0.1 to about 200 nanometers, and may also 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 also comprise buffered agents, salts, or one or more of the additional ingredients described herein. Other ophthalmically administrable formulations which are useful include those comprising the active ingredient in microcrystalline form or in a liposome preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; 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; wetting or dispersing agents; emulsifying agents, demulcentants; plug; salts; thickening agents; loads; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable hydrophobic or polymeric materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example, in Remington's, Pharmaceutical Siciences Genaro, ed., Mack Publishing Co., Easton, PA (1985). , which is incorporated herein by reference.

The active ingredient combination of the aromatase inhibitor / anti-CTLA4 of the invention may be administered to an animal, preferably a human. While the exact dosage administered of each active ingredient will vary depending on any number of factors, including but not limited to, the type of animal and type of condition being treated, the age of the animal and the routine administration. .

The anti-CTLA4 antibody may be administered to an animal as often as several times daily, or 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 once a year or less. The frequency of dosing will be readily apparent to the skilled artisan and will depend on any number of factors, such as, but not limited to, the type and severity of the disease treated, the type and age of the animal, etc.

The aromatase inhibitor, preferably exemestane, may be administered to an animal as often as several times a day, or 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 once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend on 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 aromatase may be co-administered by the fact that they may be administered separately, at different dates or at different times of the day, as well as simultaneously or on the same date. Co-administration thus encompasses any time combination of administration of the antibody and the aromatase inhibitor 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. from the other.

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

VII. Kits

The invention includes various kits comprising a therapeutically effective amount of a human anti-CTLA4 antibody of the invention and a therapeutically effective amount of an aromatase inhibitor, together with an applicator and instructional materials describing the use of the combination to perform the methods. of the invention. Although exemplary kits are described below, the contents of such useful kits will be apparent to the skilled person in light of the present disclosure. Each of these kits is included in the invention.

The invention includes a kit for treating breast cancer in a patient in need thereof. The kit includes a human anti-CTLA4 antibody of the invention and at least one aromatase inhibitor. The inhibitor abge, however, is not limited to fulvestrant, anastrozole, ietrozole and exemplan. The kit also comprises an applicator, including, but not limited to, a syringe for administering the kit components to a patient. In addition, the kit comprises instructional material presenting 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 antibody heavy and light chain amino acid sequence 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14 .3, 6.1.1, 11.2.1, 11.6, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1 (MDX-010), even more preferably, the antibody is an antibody having the amino acid sequence heavy and light chain antibodies 4.13.1, 11.2.1, and 10D1 (MDX-010).

In one embodiment, the aromatase inhibitor is at least one inhibitor selected from anastrozole, letrozole, and exemestane. More preferably, the aromatase inhibitor is exemestane.

The kit may comprise any number of additional agents for the treatment of breast cancer. Such agents are presented previously and include chemotherapeutic compounds, cancer vaccines, signal transduction inhibitors, agents useful in the treatment of cancer or abnormal cell growth, antibodies or the like that inhibit tumor growth by binding to IGF-1R, and cytokines, among many others.

The invention also relates to an article of manufacture (for example, dosage form adapted for iv administration) comprising an anti-human 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 an aromatase inhibitor. In certain embodiments, the article of manufacture comprises a container or containers comprising a human anti-CTLA4 antibody, aromatase inhibitor, and a label and / or instructions for use to treat cancer.

The invention is also described in detail with reference to the following experimental examples. These examples are provided for illustration purposes only, and are not intended to be limiting unless otherwise specified. Accordingly, the invention would in no way be construed as being limited to the following examples, but preferably should be considered to encompass any and all variations which are apparent from a result of the teaching provided herein.

EXAMPLE

Anti-CLTA4 antibody in combination with exemestane in metastatic or locally advanced breast cancer in postmenopausal patient

Patients with locally advanced or metastatic breast cancer with at least one lesion that can be measured exactly in two dimensions and whose size is> 2 cm χ 1 cm by conventional CT scan or> 1 cm χ 1 cm by spiral CT scan An IV infusion (100 ml / hr) of anti-CTLA4 antibodies was administered as described herein. Prophylactic and antidiarrheal anemetics are determined when appropriate.

The treatment is repeated after 28 days. Successive cohorts receive progressive doses of the anti-CTLA4 antibody dose, starting at 3 mg / kg and increasing to 6 mg / kg, 10 mg / kg and 15 mg / kg every 28 days, respectively, until maximum 12 cycles in the absence of intolerable toxicity or disease progression.

Preferably, patients are premedicated with antihistamine (H1) at least half an hour before anti-CTLA4 infusion. Premedication is recommended but not required.

All patients receive exemestane as a continuous oral dose of 25 mg per day of each 28-day cycle. Preferably, exemestane is taken at the same time each day.

Doses are progressive employing an accelerated titration designation using a dose doubling scheme with 3-6 subjects per cohort. In each new cohort there is no waiting period required between individuals. Subsequent cohorts cannot be opened until the first individual at the current dose level has been observed for 21 days and subsequent individuals have been observed for 14 days.

The antibody is supplied in clear 10 ml glass vials with a rubber stopper and an aluminum seal. Each vial contains 5 mg / ml (with a nominal load of 50 mg / vial) of anti-CTLA4 antibody in a sterile aqueous solution comprising 20 mM sodium acetate, 0.2 mg / ml polysorbate 80, and 140 mM of sodium chloride at pH 5.5.

For all patients, ECOG performance status, vital signs, and body weight are assessed pre-dose, and vital signs may be repeated post-dose when clinically indicated. A physical examination (including ophthalmic evaluation and autoimmunity signs) is performed on Day 1. Samples for hematology panel (hematocrit, RBC1 count differential WBC1 count), chemical (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.

The reference human anti-human antibody (HAHA) titre is determined and the pharmacokinetic specimen (PK) is obtained pre-dose.

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

Preferably, the anti-CTLA4 antibody has the heavy and light chain amino acid sequences of at least one antibody selected from 4.1.1, 4.13.1, 11.2.1, and 10D1. More preferably, the antibody has the heavy and light chain amino acid sequences of 11.2.1.

Three patients were administered anti-CTLA4 antibody (ticilimumab) at 3 mg / kg. All three patients were administered with exemestane at 25 mg daily. One patient received two doses of anti-CTLA4 every twenty-eight days and exemestane daily for 56 days. Cycle 2 stage CT revealed a lung injury not present at baseline. In addition, the patient demonstrated new thickening of the moderate circumferential wall involving the terminal ileum, distal descending colon, and sigmoid colon of uncertain etiology, possible infections or inflammatory. The patient had no clinical history to explain these changes and the patient felt well and was clinically asymptomatic. No infections were detected. This patient was out of the study for disease progression (PD).

A second patient was administered two cycles of anti-CTLA4 antibody and had approximately fourteen (14) days of exemestane therapy (25 mg daily). Following the initial ticilimumab dose, the patient reported some right arm reference value lymphedema, which made him slightly worse, then dropped. In addition, the patient reported transient moderate non-pruritic eruption.

A third patient received a ticilimumab cycle and approximately twenty-eight days of 25 mg exemestane daily until now. On day twenty-eight, routine laboratory studies detected high ALP and AST Grade 1 as well as high ALT Grade 2. The patient was asymptomatic. The therapy was put on hold until these laboratory values improved.

These data suggest that patients treated with the combination of ticilimumab and exemestane demonstrated biological activity.

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

While the invention has been described 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 considered to include all such equivalent embodiments and variations. Sequence Listing

<110> PFIZER PRODUCTS INC.

Gomez-Navarro1 Jesus

<120> CTLA4 ANTIBODY COMBINATION TREATMENT AND BREAST CANCER AROMATASE INHIBITOR

<130> PC32619A

<140> Order # to be provided

<141> 2ÕÕ5-10-07

<150> US 60 / 624,856 <151> 2004-11-04

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Phe Leu Phe Le Pro Lys Pro Lys Asp Thr Leu Met Ile Be Arg Thr 260 265 270

Pro Glu Val Thr Cys Val Val Val Asp Val Be His Glu Asp Pro Glu 275 280 285

Go Gln Phe Asn Trp Tyr Val Asp Gly Go Glu Go His Asn Ala Lys 290 295 300

Thr Lys Pro Arg Glu Glu Gln Phe Asn Be Thr Phe Arg Val Val Be 305 310 315 320

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335

Cys Lys Val Ser Asn Lys Gly Leu Pro Wing Pro lie Glu Lys Thr Ile 340 345 350

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Ser Arg Leu Glu Pro Glu Asp Phe Wing Val Tyr Tyr Cys Gln Gln Tyr 100 105 110

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Arg Thr Val Wing Ala Pro Wing Be Val Phe Ile Phe Pro Pro Asp Glu 130 135 140 Gln Leu Lys Be Gly Thr Wing Ala Val Val Cys Leu Asn Pn 145 150 155 160

Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn Wing Leu Gln 165 170 175

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Thr Tyr Be Read Be Be Thr Thr Read Read Thr Be Read Lys Wing Asp Tyr Glu 195 200 205

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Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu Pro Glu Asp Phe Ala 65 70 75 80

Val Tyr Tyr Cys Gln Gln Tyr Gly Thr Ser Pro Trp Thr Phe Gly Gln 85 90 95

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<213> Homo sapiens

<400> 13

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt agtcatggca tccactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagaaa taaagactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ttgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagtggcc 300 ccactggggc cacttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc 360 accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgctc tgaccagcgg cgtgcacacc ttcccagctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcaact tcggcaccca gacctacacc 600 tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agacagttga gcgcaaatgt 660 tgtgtcgagt gcccaccgtg cccagcacca cctgtggcag gaccgtcagt cttcctcttc 720 cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgággtcac gtgcgtggtg 780 gtggacgtga gccacgaaga ccccgaggtc cagttcaact ggtacgtgga cggcgtggag 840 gtgcataatg ccaagac aaa gccacgggag gagcagttca acagcacgtt ccgtgtggtc 900 agcgtcctca ccgttgtgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960 tccaacaaag gcctcccagc ccccatcgag aaaaccatct ccaaaaccaa agggcagccc 1020 cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc agcctgacct 1080 gcctggtcaa aggcttctac cccagcgaca tcgccgtgga 114 gtgggagagc

aatgggcagc cggagaacaa ctacaagacc acacctccca tgctggactc cgacggctcc 1201

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 126 ·

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 132

tctccgggta aatga 133

<210> 14

<211> 444

<212> PRT

<213> Homo sapiens

<400> 14

Gln Goes Gln Leu Val Glu Be Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15

Be Read Arg Read Be Cys Wing Ala Be Gly Phe Thr Phe Be Be His 20 25 30

Gly Ile His Trp Valid Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Goes 35 40 45

Val Ile Wing Trp Tyr Asp Gly Arg Asn Lys Asp Tyr Wing Asp Ser goes 50 55 60 -

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Read Tyr 65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Arg Wing Val Pro Wing Read Gly Pro Read Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Read Val Thr Val Ser Be Wing Be Thr Lys Gly Pro Ser Val Phe Pro 115 120 125

Leu Wing Pro Cys Be Arg Be Thr Be Glu Be Thr Wing Alu Leu Gly 130 135 140

Cys Leu will Lys Asp Tyr Phe Pro Glu Pro Val Thr will be Trp Asn 145 150 155 160

Be Gly Wing Leu Thr Be Gly Wing His His Phe Pro Wing Val Leu Gln 165 170 175

be be Gly Leu Tyr be Leu be Val be Thr will Pro be Ser 180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Go Asp Lys Thr Go Glu Arg Lys Cys Cys Go Glu Cys 210 215 220

Pro Pro Cys Pro Wing Pro Pro Go Gly Pro Wing Ser Val Phe Leu Phe 225 230 235 240

Pro Lys Pro Lys Pro Asp Thr Read Met Ile Ser Arg Thr Pro Glu Val 245 250 255

Thr Cys Val goes Val Asp Val Be His Glu Asp Pro Glu Val Gln Phe 260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Goes His Asn Wing Lys Thr Lys Pro 275 280 285

Arg Glu Glu Gln Phe Asn Be Thr Phe Arg Will Go Be Go Leu Thr 290 295 300

Val Goes His Gln Asp Trp Read Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320

Ser Asn Lys Gly Leu Pro Wing Pro Ile Glu Lys Thr Ile Ser Lys Thr 325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Being Arg 340 345 350

Glu Glu Met Thr Lys Asn Gln Will Be Read Thr Cys Read Val Lys Gly 355 360 365

Phe Tyr Pro Be Asp Ile Wing Val Glu Trp Glu Be Asn Gly Gln Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser 385 390 395 400

Phe Phe Read Tyr Be Lys Read Thr Val Asp Lys Be Arg Trp Gln Gln 405 410 415

Gly Asn Val Phe Be Cys Be Goes Met His Glu Wing Read His Asn His 420 425 430

Tyr Thr Gln Lys Be Read Be Read Be Pro Gly Lys 435 440

<210> 15

<211> 153

<212> PRT

<213> Homo sapiens

<400> 15

Pro Gly Arg Be Read Arg Read Le Be Cys Wing Ala Be Gly Phe Thr Phe 15 10 15 Be Be His Gly Ile His Trp Val Arg Gln Wing Pro Gly Lys Gly Leu 20 25 30

Glu Trp Val Wing Val Ile Trp Tyr Asp Gly Arg Asn Lys Asp Tyr Wing 35 40 45

Asp Be Go Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn 50 55 60

Thr Leu Tyr Leu Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Go 65 70 75 80

Tyr Tyr Cys Arg Wing Val Wing Pro Wing Read Gly Pro Wing Asp Tyr Trp Gly 85 90 95

Gln Gly Thr Read Val Thr Val Be Ser Wing Be Thr Lys Gly Pro Be 100 105 110

Go Phe Pro Read Wing Pro Cys Be Arg Be Thr Be Glu Be Thr Wing 115 120 125

Wing Read Gly Cys Read Val Lys Asp Tyr Phe Pro Glu Pro Val Thr goes 130 135 140

be Trp Asn be Gly Wing Leu Thr Ser 145 150

<210> 16

<211> 10

<212> PRT

<213> Homo sapiens

<400> 16

Gly Phe Thr Phè Being His Gly Ile His 1 5 10

<210> 17

<211> 15

<212> PRT

<213> Homo sapiens

<400> 17

Val Ile Trp Tyr Asp Gly Arg Asn Lys Asp Tyr Asp Wing Be Val 15 10 15

<210> 18

<211> 9

<212> PRT

<213> Homo sapiens

<400> 18

Val Wing Pro Read Gly Pro Read Asp Tyr 1 5

<210> 19 <211> 645 <212> DNA

<213> Homo sapiens

<400> 19

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgtcagc agctacttag cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatggt gcatccagca gggccactgg catcccagac 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct 240

gaggattttg cagtgtatta ctgtcaacag tatggtaggt caccattcac tttcggccct 300

gggaccaaag tagatatcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

<210> 20

<211> 214

<212> PRT

<213> Homo sapiens

<400> 20

Glu Ile Val Leu Thr Gln Be Pro Gly Thr Read Le Be Read Pro Gly 15 10 15

Glu Arg Wing Thr Read Be Cys Arg Wing Be Gln Be Val Ser Be Tyr 20 25 30

Leu Wing Trp Tyr Gln Lys Pro Gly Gln Wing Pro Arg Leu Leu Ile 35 40 45

Tyr Gly Wing Ser Be Arg Wing Thr Gly Ile Pro Asp Arg Phe Be Gly 50 55 60

be Gly Be Gly Thr Asp Phe Thr Leu Thr Ile be Arg Leu Glu Pro 65 70 75 80

Glu Asp Phe Ala Go Tyr Tyr Cys Gln Gln Tyr Gly Arg Be Pro Phe 85 90 95

Thr Phe Gly Pro Gly Thr Lys Go Asp Ile Lys Arg Thr Val Wing Wing 100 105 110

Pro to be Val Phe lie Phe Pro to be Asp Glu Gln Read Lys be Gly 115 120 125

Thr Wing Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Wing 130 135 140

Lys Val Gln Trp Lys Val Asp Asn Wing Read Gln Be Gly Asn Be Gln 145 150 155 160

Glu Be Val Thr Glu Gln Asp Be Lys Asp Be Thr Tyr Be Leu Be 165 170 175

Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Goes Thr His Gln Gly Leu Ser Ser Pro Goes Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

<210> 21

<211> 146

<212> PRT

<213> Homo sapiens

<400> 21

Gln Be Pro Gly Thr Read Be Be Read Pro Gly Glu Arg Wing Thr Thru 1 5 10 15

Be Cys Arg Wing Be Gln Be Val Ser Be Tyr Leu Wing Trp Tyr Gln 20 25 30

Gln Lys Pro Gly Gln Pro Wing Arg Read Leu Ile Tyr Gly Wing Ser Ser 35 40 45

Arg Wing Thr Gly Ile Pro Asp Arg Phe Be Gly Be Gly Be Gly Thr 50 55 60

Asp Phe Thr Leu Thr Ile be Arg Leu Glu Pro Glu Asp Phe Wing Val 65 70 75 80

Tyr Tyr Cys Gln Tyr Gly Arg Be Pro Phe Thr Phe Gly Pro Gly 85 90 95

Thr Lys Go Asp Ile Lys Arg Thr Val Wing Ward Pro Ser Val Phe Ile 100 105 110

Phe Pro Pro Be Asp Glu Gln Read Lys Be Gly Thr Wing Be Val Val 115 120 125

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys 130 135 140

go Asp 145

<210> 22

<211> 11

<212> PRT

<213> Homo sapiens <400> 22

Arg Wing Be Gln Be Val Be Ser Tyr Leu Wing 15 10

<210> 23

<211> 7

<212> PRT

<213> Homo sapiens

<400> 23

Gly Wing Be Ser Arg Wing Thr 1 5

<210> 24

<211> 10

<212> PRT

<213> Homo sapiens

<400? · 24

Cys Gln Gln Tyr Gly Arg Ser Phe Pro 1 5 10

<210> 25

<211> 1413

<212> DNA

<213> Homo sapiens

<400> 25

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagtagc tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagttata tggtatgatg gaagtaataa atactatgca 240 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag agatccgagg 360 ggagctaccc tttactacta ctactacggt atggacgtct ggggccaagg gaccacggtc 420 accgtctcct cagcctccac caagggccca tcggtcttcc ccctggcgcc ctgctccagg 480 agcacctccg agagcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540 gtgacggtgt cgtggaactc aggcgctctg accagcggcg tgcacacctt cccagctgtc 600 ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcaacttc 660 ggcacccaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 720 acagttgagc gcaaatgttg tgtcgagtgc ccaccgtgcc cagcaccacc tgtggcagga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacgt gcgtg gtggt ggacgtgagc cacgaagacc ccgaggtcca gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cacgggagga gcagttcaac 960 agcacgttcc gtgtggtcag cgtcctcacc gttgtgcacc aggactggct gaacggcaag 1020 gagtacaagt gcaaggtctc caacaaaggc ctcccagccc ccatcgagaa aaccatctcc 1080 aaaaccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc 1200 cagcgacatc

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac acctcccatg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tccgggtaaa tga 1413

<210> 26

<211> 451

<212> PRT

<213> Homo sapiens

<400> 26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15

Be Read Arg Read Be Cys Wing Wing Be Gly Phe Thr Phe Be Ser Tyr 20 25 30

Gly Met His Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val 35 40 45

Val Ile Wing Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Read Tyr 65 70 75 80

Read Gln Met Asn Be Read Arg Wing Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Arg Wing Asp Pro Arg Gly Wing Thr Read Tyr Tyr Tyr Tyr Gyr Met 100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Be Wing Ser Thr 115 120 125

Lys Gly Pro To Be Val Phe Pro To Read Wing Pro Cys To Be Arg To Thr Be 130 135 · 140

Glu Ser Thr Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe Pro Glu 145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His 165 170 175

Thr Phe Pro Wing Val Leu Gln Be Gly Leu Tyr Be Read Ser Be 180 185 190

Go Go Thr Go Pro Be Asn Phe Gly Thr Gln Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Be Asn Thr Lys Val Asp Lys Thr Go Glu 210 215 220

Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Wing Pro Pro Val Wing 225 230 235 240

Gly Pro Ser will Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met 245 250 255

Ile Ser Arg Thr Pro Glu Go Thr Cys Go Val Val Asp Val Be His 260 265 270

Glu Asp Pro Glu Goes Glri Phe Asn Trp Tyr Val Asp Gly Val Glu Goes 275 280 285

His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Be Thr Phe 290 295 300

Arg Val will be will Leu Thr Val Val His Gln Asp Trp Leu Asn Gly 305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Pro Wing Ile 325 330 335

Glu Lys Thr Ile Be Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln goes 340 345 350

Tyr Thr Leu Pro Pro Be Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Wing Val Glu 370 375 380

Trp Glu Be Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400

Met Leu Asp Be Asp Gly Be Phe Phe Leu Tyr Be Lys Leu Thr Val 405 410 415

Asp Lys Be Arg Trp Gln Gln Gly Asn Val Phe Be Cys Be Val Met 420 425 430

His Glu Ala Read His Asn His Tyr Thr Gln Lys Be Read Be Read 435 440 445

Pro Gly Lys 450

<210> 27

<211> 167

<212> PRT

<213> Homo sapiens

<400> 27 Gly Goes Go Gln Pro Gly Arg Be Leu Arg Leu Be Cys Wing Wing Be 1 5 10 15

Gly Phe Thr Phe Be Tyr Gly Met His Trp Val Arg Gln Wing Pro 20 25 30

Gly Lys Gly Leu Glu Trp Val Wing Val Ile Trp Tyr Asp Gly Ser Asn 35 40 45

Lys Tyr Tyr Wing Asp Be Goes Lys Gly Arg Phe Thr Ile Be Arg Asp 50 55 60

Asn Be Lys Asn Thr Read Tyr Read Gln Met Asn Be Read Arg Wing Glu 65 70 75 80

Asp Thr Wing Val Tyr Tyr Cys Arg Wing Asp Pro Arg Gly Wing Thr 85 90 95

Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110

Thr Will Be Sera Wing Be Thr Lys Gly Pro Ser Will Phe Pro Leu Wing 115 120 125

Pro Cys Be Arg Be Thr Be Glu Be Thr Ward Wing Read Gly Cys Read 130 130 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr will be Trp Asn be Gly 145 150 155 160

Wing Read Thr Be Gly Val His 165

<210> 28 <211> 10 <212> PRT <213> Homo sapiens

<400> 28

Gly Phe Thr Phe Being Ser Tyr Gly Met His 1 5 10

<210> 29

<211> 15

<212> PRT

<213> Homo sapiens

<400> 29

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Wing Asp Ser Val 1 5 10 15

<210> 30

<211> 16

<212> PRT

<213> Homo sapiens <400> 30

Asp Pro Arg Gly Wing Thr Read Tyr Tyr Tyr Tyr Gyr Met Asp Val 15 10 15

<210> 31

<211> 714

<212> DNA

<213> Homo sapiens

<400> 31

atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60

agatgtgaca tccagatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgccgggc aagtcagagc attaacagct atttagattg gtatcagcag 180

aaaccaggga aagcccctaa actcctgatc tatgctgcat ccagtttgca aagtggggtc 240

ccatcaaggt tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 300

caacctgaag attttgcaac ttactactgt caacctgaag actagtactcc attcactttc 360

ggccctggga ccaaagtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta gtga 714

<210> 32

<211> 214

<212> PRT

<213> Homo sapiens

<400> 32

Asp Xle Gln Met Thr Gln Be Pro Be Be Read Be Wing Be Be Gly 1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Wing Be Gln Ser Lie Asn Ser Tyr 20 25 30

Read Asp Trp Tyr Gln Gln Lys Pro Gly Lys Wing Pro Lys Leu Leu Ile 35 40 45

Tyr Wing Wing Ser Be Read Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80

Glu Asp Phe Wing Thr Thr Tyr Cys Gln Gln Tyr Tyr Be Thr Pro Phe 85 90 95

Thr Phe Gly Pro Gly Thr Lys Go Glu Ile Lys Arg Thr Val Wing Wing 100 105 110 Pro Be Val Phe Ile Phe Pro Pro Be Asp Glu Leu Lys Ser Gly 115 120 125

Thr Wing will be Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Wing 130 135 140

Lys Val Gln Trp Lys Val Asp Asn Wing Read Gln Ser Gly Asn Ser Gln 145 150 155 160

Glu Be Go Thr Thr Glu Gln Asp Be Lys Asp Be Thr Tyr Be Read 165 165 175

Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala cys Glu Val Thr His Gln Gly Read Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210

<210> 33

<211> 139

<212> PRT

<213> Homo sapiens

<400> 33

Pro Be Be Leu Be Wing Be Val Gly Asp Arg Go Thr Ile Thr Cys 15 10 15

Arg Wing Be Gln Be Ile Asn Be Tyr Read Asp Trp Tyr Gln Gln Lys 20 25 30

Pro Gly Lys Wing Pro Lys Leu Read He Tyr Wing Al Ser Be Leu Gln 35 40 45

Be Gly Go Pro Be Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe 50 55 60

Thr Leu Thr Ie Ser Be Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr 65 70 75 80

Cys Gln Gln Tyr Tyr Be Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 85 90 95

Go Glu Ile Lys Arg Thr Val Wing Ward Pro Be Val Phe Ile Phe Pro 100 105 110

Pro be Asp Glu Gln Leu Lys Ser Gly Thr Wing Ser will Val cys Leu 115 120 125

Read Asn Asn Phe Tyr Pro Arg Glu Ala Lys goes 130 135 <210> 34

<211> 11

<212> PRT

<213> Homo sapiens

<400> 34

Arg Wing Be Gln Be Ile Asn Be Tyr Leu Asp 5 10

<210> 35

<211> 7

<212> PRT

<213> Homo sapiens

<400> 35

Wing Wing Ser Ser Leu Gln Ser 1 5

<210> 36

<211> 9

<212> PRT

<213> Homo sapiens

<400> 36

Gln Gln Tyr Tyr Ser Thr Pro Phe Thr 1 5

Claims (20)

Method for the treatment of breast cancer in a patient in need of such treatment, characterized in that it comprises administering to said patient a therapeutically effective amount of an anti-CTLA4 antibody, or antigen binding portion thereof, in combination with a therapeutically effective amount of an aromatase inhibitor. A method according to claim 1, characterized in that said aromatase inhibitor is at least one inhibitor selected from the group consisting of anastrozole, letrozole, and exemplan. Method according to claim 2, characterized in that said aromatase inhibitor is exemestane. The method of claim 3, wherein said therapeutically effective amount of an amount of human anti-CTLA4 antibody ranges from about 1 mg / kg to 40 mg / kg. The method of claim 4, wherein said therapeutically effective amount of an amount of human anti-CTLA4 antibody ranges from about 3 mg / kg to 15 mg / kg. Method according to claim 3, characterized in that said therapeutically effective amount of urestane ranges from about 25 mg per day to 200 mg per day. Method according to claim 6, characterized in that said therapeutically effective amount of exestan is about 25 mg per day. A method according to claim 1, wherein said treatment is selected from the group consisting of neoadjuvant therapy, adjuvant therapy, first line treatment, second line treatment, and a third line treatment. A method according to claim 1, wherein said antibody is selected from the group consisting of a non-human mammalian antibody, a chimeric antibody, and a human antibody. Method according to claim 9, characterized in that said antibody is a human antibody. A method according to claim 1, wherein said anti-CTLA4 antibody, or antigen binding portion thereof, is at least one antibody selected from the group consisting of: (a) a human antibody which has a CTLA4 binding affinity of about 10-8 or greater, and inhibits binding between CTLA4 and B7-1, and binding between CTLA4 and B7-2; (b) a human antibody that has an amino acid sequence. an acid comprising at least one human CDR sequence corresponding to a CDR sequence 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, 11.6.1, -11.7.1., 12.3.1.1, 12.9.1.1, and 10D1; (c) a human antibody that has the heavy and light chain amino acid sequences of a selected antibody 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, 11.6.1, 11.7.1., -12.3.1.1, and 12.9. 1.1; (d) a human antibody which has the sequences of a minoxide of a heavy chain variable region and a light chain variable region 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1; (e) an antibody, or antigen binding portion thereof, that competes for binding to CTLA4 with at least one 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, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1; and (f) an antibody, or antigen binding portion thereof, which interacts for CTLA4 binding with at least one antibody which has the heavy and light chain amino acid sequences of an antibody selected from the group that consists of 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, -11.2.1, 11.6.1, 11.7.1., 12.3.1.1, 12.9.1.1, and 10D1 . A method according to claim 1, characterized in that said antibody is a human antibody having the antibody heavy and light chain amino acid sequences. 11.2.1. A method according to claim 1, characterized in that said antibody comprises a heavy chain and a light chain wherein the amino acid sequences of the heavy chain variable region of said heavy chain and the light chain variable region from said light chain are selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 9; (b) the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 21; (c) the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 33; (d) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 1 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 7; (e) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 13 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 19; (f) the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 25 and the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 31; (g) in the amino acid sequence of a heavy chain variable region and a 10D1 antibody light chain variable region. A method according to claim 1, wherein said antibody, or antigen binding portion thereof, is an antibody selected from the group consisting of: (a) an antibody having a heavy chain variable region which comprises the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and also having a light chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 10 SEQ ID NO: 11 and SEQ ID NO: 12; (b) an antibody having a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and also having a light chain variable region which comprises the amino acid sequences shown SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; (c) an antibody having a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, and also having a light chain variable region which comprises the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; and (d) an antibody having a heavy chain variable region comprising the 10D1 antibody heavy chain amino acid sequences CDR1, CDR2, and CDR3, also having a light chain variable region comprising the light chain amino acid sequences CDR1, CDR2, and CDR3 of 10D1 antibody. The method of claim 1 further comprising administering to said patient at least one agent selected from the group consisting of an alkylating agent, a folate antagonist, a pyrimidine antagonist, an anthracillin antibiotic, a platinum compound, a taxane, an alkaloid vinca, a camptothecin analog, a toll-like receptor stimulating agent, a heat shock protein-based tumor vaccine, an antigen-presenting cell-based therapy, a rapamycin inhibitor mammalian target, an erbB2 inhibitor, an EGFR inhibitor, a VEGF inhibitor, a VEGFR inhibitor, an angiogenesis inhibitor, an antibody, an immunomodulator, a selective estrogen receptor modulator, a cytokine, a tumor vaccine, an antiproliferative agent, an immune costimulatory molecule, and a cytokine. A pharmaceutical composition for the treatment of breast cancer, comprising: a therapeutically effective amount of an anti-CTLA4 antibody; a therapeutically effective amount of a flavor inhibitor; and a pharmaceutically acceptable carrier. Pharmaceutical composition according to claim 16, characterized in that said aromatase inhibitor is at least one aromatase inhibitor selected from the group consisting of anastrozole, etrozole and exemestane. Pharmaceutical composition according to claim 17, characterized in that said aromatase inhibitor is exemplary. 19. Use of an amount of an anti-CTLA4 antibody, characterized in that it is in the preparation of a composition for the treatment of breast cancer in a patient wherein said treatment further comprises administering to said patient. an amount of an aromatase inhibitor. Use according to claim 19, characterized in that said aromatase inhibitor is exemestane and also wherein said antibody is a human antibody having the heavy and light chain variable region amino acid sequences. of antibody 11.2.1.