Anti-CTLA4 antibodies, antibody fragments, their immunoconjugates and uses thereof

Abstract

To provide anti-CTLA4 antibodies.SOLUTION: Anti-CTLA4 antibodies or antibody fragments comprising a heavy chain variable region having a specific amino acid sequence and a light chain variable region having a specific amino acid sequence, an immunoconjugate comprising the antibodies or antibody fragments, and a pharmaceutical composition comprising the immunoconjugate are provided.SELECTED DRAWING: Figure 3A

Description

Technical Field

[0001] The present disclosure relates to anti-CTLA4 antibodies, antibody fragments, and immunoconjugates of such antibodies and antibody fragments, and to the use of antibodies, antibody fragments, and immunoconjugates in diagnostic and therapeutic methods.

Background Art

[0002] The immune system of vertebrates requires multiple signals to achieve optimal immune activation. See, for example, Janeway, Cold Spring Harbor Symp. Quant. Biol. 54:1-14 (1989); Paul William E., ed. Raven Press, N.Y., Fundamental Immunology, 4th edition (1998), particularly, see pages 12, 13, and 411-478. The interaction between T lymphocytes (T cells) and antigen-presenting cells (APCs) is essential for immune activation. The levels of many adhesion molecules found on T cells and APCs increase during immune activation (Springer et al., A. Rev. Immunol., 5:223-252 (1987), Shaw and Shimuzu, Current Opinion in Immunology, Eds. Kindt and Long. 1:92-97 (1988)), and Hemler, Immunology Today, 9:109-113 (1988)). The increase in the levels of these molecules can help explain why activated APCs are more effective than resting APCs in stimulating antigen-specific T cell proliferation (Kaiuchi et al., J. Immunol., 131:109-114 (1983), Krieger et al., J. Immunol., 135:2937-2945 (1985), McKenzie, J. Immunol., 141:2907-2911 (1988), and Hawrylowicz and Unanue, J. Immunol., 141:4083-4088 (1988)).

[0003] T cell immune responses are complex processes involving cell-cell interactions, particularly between T cells and accessory cells such as APCs (Springer et al., A.Rev.Immumol., 5:223-252 (1987)), as well as the production of soluble immune mediators (cytokines or lymphokines) (Dinarello, New Engl.Jour: Med., 317:940-945 (1987), Sallusto, J.Exp.Med., 179:1109-1118 (1997)). Immune responses are regulated by several T cell surface receptors, including the T cell receptor complex (Weiss, Ann.Rev.Immunol., 4:593-619 (1986)), and other "accessory" surface molecules (Allison, Curr.Opin.Immunol., 6:414-419 (1994); Springer (1987), supra). Many of these accessory molecules are naturally occurring cell surface differentiation antigens defined by the reactivity of monoclonal antibodies on the cell surface (McMichael, Ed., Leukocyte Tiping III, Oxford Univ.Press, Oxford, N.Y. (1987)).

[0004] CTLA4 is a T cell surface molecule originally identified by differential screening of a cDNA library of murine cytotoxic T cells (Brunet et al., Nature 328:267-270 (1987)). CTLA4 is also a member of the immunoglobulin (Ig) superfamily. CTLA4 contains a single extracellular Ig domain. CTLA4 transcripts have been found in T cell populations with cytotoxic activity, and it has been found that CTLA4 can function in the cytotoxic response (Brunet et al. (supra); Brunet et al., Immunol. Rev., 103:21-36 (1988)). The researchers reported the cloning of the gene and mapping to the same chromosomal region (2d, 33-34) as CD28 for the human counterpart of CTLA4 (Dariavach et al., Eur: J. Immunol., 18:1901-1905 (1988)), CD28 (Lafage-Pochitaloff et al. Immunogenetics, 31:198-201 (1990)). Sequence comparison between this human CTLA4 DNA and the DNA encoding the CD28 protein revealed significant sequence homology, with the greatest homology in the region near the membrane and the cytoplasmic region (Brunet et al., 1988 (supra), Dariavach et al., 1988 (supra)).

[0005] Several studies have suggested that CTLA4 has a similar function as a secondary costimulatory factor (Linsley et al., J. Exp. Med., 176:1595-1604 (1992), Wu et al., J. Exp. Med., 185:1327-1335 (1997), and U.S. Pat. Nos. 5,977,318, 5,968,510, 5,885,796, and 5,885,579). However, others have reported that CTLA4 has an opposing role as an inhibitor of T cell activation (Krummel, J. Exp. Med., 182:459-465 (1995), Krummel et al., Int’l Immunol., 8:519-523 (1996), Chambers et al., Immunity, 7:885-895 (1997)). CTLA4-deficient mice have been reported to suffer from massive lymphoproliferation (Chambers et al., supra). Also, blockade of CTLA4 has been reported to enhance T cell responses and exacerbate antitumor immunity (Leach, Science, 271:1734-1736 (1996)) and enhance induced autoimmune diseases (Luhder, J Exp. Med., 187:427-432 (1998)) in vitro (Walunas et al., Immunity, 1:405-413 (1994)) and in vivo (Kearney, J. Immunol., 155:1032-1036 (1995)). CTLA4 has also been reported to have an alternative or additional effect on the early characteristics of T cell immune responses (Chambers, Curr. Opin. Immunol., 9:396-404 (1997), Bluestone, J. Immunol., 158:1989-1993 (1997), Thompson, Immunity, 7:445-450 (1997)). This is consistent with the observation that some autoimmune patients have autoantibodies against CTLA4. Blocking antibodies to CTLA4 may have a pathogenic role in these patients (Matsui, J. Immunol., 162:4328-4335 (1999)).

[0006] CTLA4 has been shown to negatively regulate immune activation through both endogenous and exogenous mechanisms. See Grosso and Kunkel, Cancer Immunity, 13:5 (2013). Specifically, (i) reverse signaling through CD80 and CD86 on APCs suppresses T cell responses and / or promotes the conversion of naive T cells to Tregs, (ii) signaling through CTLA3 stimulates the production of regulatory cytokines such as TGF-β, resulting in inhibition of antigen presentation by APCs and inhibition of T cell function, (iii) when CTLA4 binds to CD80 / CD86, the availability of ligands for binding by CD28 is reduced, (iv) when CTLA4 binds to CD80 / CD86, it causes their transendocytosis, reducing the ability of APCs to activate T cells, (v) CTLA4 recruits inhibitory proteins such as PP2A and PTPN11 to the T cell synapse, inhibiting signaling through CD28 and TCR, (vi) CTLA4 acts as a high-affinity competitor that occupies CD80 / 86, thereby preventing binding by CD28, (vii) soluble splice variants of CTLA4 may be able to inhibit T cell activation, (viii) CTLA4 inhibits the T cell stop signal important for activation of T cells by APCs.

[0007] Therefore, inhibition of CTLA4 has been shown to promote the stimulation of adaptive immune responses and T cell activation. CTLA4 blocking antibodies have been shown to be effective in mouse models of cancer, and anti-CTLA4 antibodies such as ipilimumab (WO2001 / 014424) and tremelimumab are being investigated as strategies for promoting antitumor immunity in cancer. Blockade of CTLA4 is also a promising therapeutic strategy for disorders associated with T cell exhaustion such as chronic viral infection.

[0008] Antibodies against CTLA4 have been previously developed. U.S. Patent No. 9,758,583 discloses antibodies or antibody fragments that are said to bind to one or both of human and mouse CTLA4 and can be formulated into compositions for the treatment of cancer. Some of the antibodies or antibody fragments are also said to inhibit or prevent the interaction or functional association between human CTLA4 and human CD80 or CD86, or between mouse CTLA4 and mouse CD80 or CD86. Such inhibition or prevention of the interaction or functional association between CTLA4 and CD80 or CD86 may inhibit or prevent CD80- or CD86-mediated activation of CTLA4, CD80 / CTLA4 signaling, or CD86 / CTLA4 signaling.

[0009] Also, US2009 / 0252741 discloses monoclonal antibodies that bind to human CTLA4. These anti-CTLA4 antibodies are said to induce protection against cancer and also exhibit some autoimmune side effects. The antibodies that induced the strongest protection against cancer also induced the least autoimmune side effects. US2009 / 0252741 also provides a method for selecting an optimal anti-CTLA4 antibody or other therapeutic agent that has the most desirable balance between cancer protection and autoimmune side effects.

[0010] US2016 / 0237154 discloses compositions and methods related to or derived from anti-CTLA4 antibodies or antibody fragments. The anti-CTLA4 antibodies and antibody fragments can block the binding of human CTLA4 to human B7 and are thus said to be suitable for the treatment of prostate cancer, kidney cancer, colon cancer, lung cancer, or breast cancer, pathogenic infections, diseases related to the central nervous system, such as amyloidogenic diseases including Alzheimer's disease, and diseases having an inflammatory or allergic component such as graft-versus-host disease, host-versus-graft disease, allergies, autoimmune diseases, and other inflammatory diseases.

[0011] Antibodies against CTLA4 are known and commercially available, but it is desirable to find improved anti-CTLA4 antibodies suitable for cancer therapies with reduced or minimal side effects. The present invention provides, in particular, anti-CTLA4 antibodies or antibody fragments suitable for therapeutic and diagnostic use for the diagnosis and treatment of cancer. Some of these anti-CTLA4 antibodies or antibody fragments may have a higher binding affinity for CTLA4 in tumors compared to CTLA4 present in normal tissues. These anti-CTLA4 antibodies or antibody fragments typically have at least equivalent efficacy to known anti-CTLA4 antibodies or antibody fragments. In addition, the anti-CTLA4 antibodies or antibody fragments of the present invention may exhibit reduced side effects compared to monoclonal anti-CTLA4 antibodies known in the art. These advantages can provide more selective treatment of CTLA4 in tumors, and as a result of the selectivity for CTLA4 in tumors, it may be possible to use higher doses of these anti-CTLA4 antibodies or antibody fragments, thereby enabling more effective therapeutic treatment without a corresponding increase in undesirable side effects.

Summary of the Invention

[0012] In one aspect, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to the CTLA4 protein. These polypeptides include three complementarity determining regions having H1, H2, and H3 sequences, The H1 sequence is GFTFSHYTMH (SEQ ID NO: 1), The H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (SEQ ID NO: 2), The H3 sequence is TGWLGPFDX8 (SEQ ID NO: 3), wherein X1 is S or D, X2 is D, H or I, X3 is N or Y, X4 is Y or I, X5 is Y or E, X6 is D or K, X7 is V or M, and X8 is Y or I.

[0013] In another aspect, the invention includes a product formed by a combination of any one of the isolated heavy chain variable region polypeptides described above and an isolated light chain variable region polypeptide selected from isolated light chain variable region polypeptides that include three complementarity determining regions having L1, L2, and L3 sequences, The L1 sequence is RX9SQX 10 X 11 is GSSYLA (SEQ ID NO: 4), The L2 sequence is GAFSRATGX 12 (SEQ ID NO: 5), The L3 sequence is QQDGSSPWT (SEQ ID NO: 6), wherein X9 is A or I, X 10 is Y, S or H, X 11 is V or G, X 12 is V or I.

[0014] In each of the foregoing embodiments, the H2 sequence can be selected from FIDYHGNNKYYADSVKG, FISYDGNNKIYADSVKG, FISYDGNNKYYADSVKG, FISYDGNYKYYADSVKG, FISYDGNYKYYAKSVKG, FISYHGNNKYEADSVKG, FISYHGNNKYYADSVKG, FISYIGNYKYYADSMKG, and FISYIGNYKYYADSVKG.

[0015] In each of the foregoing embodiments, the H3 sequence can be selected from TGWLGPFDY and TGWLGPFDI.

[0016] In each of the foregoing embodiments, the L1 sequence can be selected from RASQHVGSSYLA, RASQSVGSSYLA, RASQYGGSSYLA, RASQYVGSSYLA, and RISQYVGSSYLA.

[0017] In each of the foregoing embodiments, the L2 sequence can be selected from GAFSRATGI and GAFSRATGV.

[0018] In some embodiments, the isolated heavy chain variable region polypeptide may have a sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38. In each of these embodiments, the isolated light chain variable region polypeptide may have a sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.

[0019] In one embodiment, the antibody comprises a light chain variable region polypeptide and a heavy chain variable region polypeptide, and has a pair of sequences selected from the following pairs: SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, SEQ ID NO: 31 and 32, SEQ ID NO: 33 and 34, SEQ ID NO: 35 and 36, and SEQ ID NO: 37 and 38.

[0020] In yet another aspect, the present invention provides an anti-CTLA4 antibody or antibody fragment comprising any one of the isolated heavy chain variable region polypeptides of the present invention described above.

[0021] In yet another aspect, the present invention provides an anti-CTLA4 antibody or antibody fragment comprising a combination of any one of the isolated heavy chain variable region polypeptides of the present invention described above and any one of the isolated light chain variable region polypeptides of the present invention described above.

[0022] In yet another aspect, the present invention provides an immunoconjugate comprising any one of the antibodies or antibody fragments of the present invention described above. In the immunoconjugate, the antibody or antibody fragment may be conjugated to a drug selected from chemotherapeutic agents, radioisotopes, cell division inhibitors, and cytotoxic agents.

[0023] In yet another aspect, the present invention provides a pharmaceutical composition comprising, together with a pharmaceutically acceptable carrier, any one of the polypeptides, antibodies, antibody fragments, and immunoconjugates of the present invention described above. The single dose of the pharmaceutical composition may contain an amount of polypeptide, antibody, antibody fragment, or immunoconjugate of about 135 mg, about 235 mg, about 335 mg, about 435 mg, about 535 mg, about 635 mg, about 735 mg, about 835 mg, about 935 mg, about 1035 mg, about 1135 mg, about 1235 mg, or about 1387 mg.

[0024] The single dose of the pharmaceutical composition may contain an amount of polypeptide, antibody, antibody fragment, or immunoconjugate in the range of 135 - 1387 mg, 135 - 235 mg, 235 - 335 mg, 335 - 435 mg, 435 - 535 mg, 535 - 635 mg, 635 - 735 mg, 735 - 835 mg, 835 - 935 mg, 935 - 1035 mg, 1035 - 1135 mg, 1135 - 1235 mg, or 1235 - 1387 mg.

[0025] Each of the aforementioned pharmaceutical compositions may further comprise an immune checkpoint inhibitor molecule different from the polypeptide or antibody or antibody fragment. The immune checkpoint inhibitor molecule can be an antibody or antibody fragment against an immune checkpoint. The immune checkpoint can be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4 - 1BB, PD - 1, PD - L1, GITR, B7 - H3, B7 - H4, KIR, A2aR, CD27, CD70, DR3, and ICOS, or the immune checkpoint can be PD - 1 or PD - L1.

[0026] Each of the foregoing pharmaceutical compositions may further comprise an antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins. The WNT protein may be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

[0027] In yet another aspect, the present invention provides a kit for diagnosis or treatment, the kit comprising any one of the polypeptides, antibodies, antibody fragments, or immunoconjugates of the present invention described above.

[0028] In yet another aspect, the present invention provides an anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 39-41, and the light chain variable region comprising three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 42-44.

[0029] In the foregoing embodiments, the heavy chain variable region may have the amino acid sequence of SEQ ID NO: 8, and the light chain variable region may have the amino acid sequence of SEQ ID NO: 7. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figures 9A - 9F

Figures 10A - 10F

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 13A

Figure 13B

Figure 14A

Figure 14B

[0031] Definitions To facilitate the understanding of the examples provided herein, certain frequently occurring terms are defined here.

[0032] In relation to the measured amount, the term "about" as used herein refers to the normal variation in the amount that would be predicted by one of ordinary skill in the art making the measurement and exercising reasonable care commensurate with the purpose of the measurement and the precision of the measuring device used. Unless otherwise indicated, "about" refers to a variation of + / - 10% of the value provided.

[0033] As used herein, the term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, when used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0034] As used herein, the term "affinity matured" antibody refers to an antibody having one or more modifications in one or more complementarity determining regions, and such modifications result in an improvement in the affinity of the antibody for the antigen as compared to the parental antibody that does not have such modifications.

[0035] As used herein, the term "amino acid" refers to any organic compound containing an amino group (-NH2) and a carboxyl group (-COOH), preferably either as a free radical or alternatively after condensation as part of a peptide bond. "α-Amino acids that form the 20 naturally encoded polypeptides" are understood in the art and refer to alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0036] As used herein, the term "antibody" refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules that can bind to an epitope of an antigen, e.g., Fab, Fab’, (Fab’)2, Fv, and SCA fragments. These antibody fragments that retain some ability to selectively bind to the antigen of the antibody from which they are derived (e.g., a polypeptide antigen) can be made using methods well known in the art (see, e.g., Harlow and Lane, supra) and are further described below. Antibodies can be used to isolate amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or staging of diseases (e.g., tumor formation), as well as for therapeutic applications for treating diseases (e.g., tumor formation, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, etc.). Chimeric antibodies, human-like antibodies, humanized antibodies, or fully human antibodies are particularly useful for administration to human patients.

[0037] The Fab fragment consists of the monovalent antigen-binding fragment of the antibody molecule and is produced by digesting the whole antibody molecule with the enzyme papain, and a fragment consisting of an intact light chain and a part of the heavy chain can be obtained.

[0038] The Fab' fragment of the antibody molecule can be obtained by treating the whole antibody molecule with pepsin and then reducing it, and a molecule consisting of an intact light chain and a part of the heavy chain can be obtained. In this manner, two Fab' fragments are obtained per treated antibody molecule.

[0039] The (Fab')2 fragment of the antibody can be obtained by treating the whole antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0040] The Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0041] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that includes a part of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0042] As used herein, the terms "anti-CTLA4 antibody", "CTLA4 antibody", and "antibody that binds to CTLA4" refer to antibodies that can bind to CTLA4 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CTLA4. In one embodiment, the degree of binding of the anti-CTLA4 antibody to an unrelated non-CTLA4 protein is less than about 10% of the binding of the antibody to CTLA4 when measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CTLA4 has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-CTLA4 antibody binds to an epitope of CTLA4 that is conserved among CTLA4s from different species.

[0043] As used herein, the term "binding" refers to the interaction of the variable region or Fv of an antibody with an antigen that has an interaction in response to the presence of a specific structure (e.g., antigenic determinant or epitope) on the antigen. For example, the variable region or Fv of an antibody generally recognizes and binds to the structure of a specific protein rather than to the protein per se. As used herein, the terms "specifically binding" or "binding specifically" mean that the antibody variable region or Fv binds or associates with a particular antigen more frequently, rapidly, for a longer duration, and / or with higher affinity than with other proteins. For example, the variable region or Fv of an antibody specifically binds to that antigen with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other antigens. In another example, the variable region or Fv of an antibody binds to a cell surface protein (antigen) with a substantially higher affinity than to related proteins or other cell surface proteins that are generally recognized by polyreactive natural antibodies (i.e., natural antibodies known to bind to various antigens naturally found in humans), or than to the affinity for the antigen. However, "specifically binding" does not necessarily require exclusive binding or undetectable binding to another antigen, which is meant by the term "selective binding". In one example, "specific binding" of the variable region or Fv of an antibody (or other binding region) means that the variable region or Fv of the antibody binds to the antigen with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, such as 20 nM or less, such as 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0044] As used herein, the terms "cancer" and "cancerous" typically refer to or describe a physiological state in a mammal characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0045] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0046] As used herein, the term "chemotherapeutic agent" refers to chemical substances useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, ureidopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bradykin and bradykinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); betalapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, for example, Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor;Dynemicin including dynemicin A; esperamicin; and neocarzinostatin chromophore and related chromoprotein engyin antibiotic chromophores), actinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN (registered trademark), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL (registered trademark)), liposome doxorubicin TLC D-99 (MYOCET (registered trademark)), pegylated liposome doxorubicin (CAELYX (registered trademark), and including deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rhodomycin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR (registered trademark)), tegafur (UFTORAL (registered trademark)), capecitabine (XELODA (registered trademark)), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; adrenocorticolytic agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;Demecolcine; Diacontin; Elfornithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triacontin; 2,2’,2’-Trichlorotriethylamine; Trichothecene (especially, T-2 toxin, verracurin A, loridin A, anguidine); Urethane; Vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)); Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gasitocin; Arabinoside (「Ara-C」); Thiotepa; Taxoids, for example, paclitaxel (TAXOL (registered trademark)), albumin-modified nanoparticle formulation of paclitaxel (ABRAXANE (trademark)), and docetaxel (TAXOTERE (registered trademark)); Chlorambucil; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum agents such as cisplatin, oxaliplatin (for example, ELOXATIN (registered trademark)), and carboplatin; Vinca that prevents the formation of microtubules by tubulin polymerization such as vinblastine (VELBAN (registered trademark)), vincristine (ONCOVIN (registered trademark)), vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)), vinorelbine (NAVELBINE (registered trademark)); Etoposide (VP-16); Ifosfamide; Mitoxantrone; Leucovorin; Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMF (registered trademark)); Retinoids such as retinoic acid including bexarotene (TARGRETIN (registered trademark));Bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY 43-9006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); olaphnib, ABT-510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine / threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; combinations of two or more of the above, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisone);And FOLFOX (an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN (trademark)) combined with 5-FU and leucovorin);

[0047] As defined herein, chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth. They may themselves be hormones, including, but not limited to, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3, anti-estrogens having a mixed agonist / antagonist profile; pure anti-estrogens without agonist properties such as fulvestrant (FASLODEX®) and EM800 (agents that can block dimerization of estrogen receptor (ER), inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors such as anastrozole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide, and other aromatase inhibitors including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and triptorelin; progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and Premarin, sex steroids including androgen / retinoids such as fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; anti-progesterone; estrogen receptor downregulators (ERD); anti-androgens such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above are included.

[0048] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species and the remainder of the heavy and / or light chain is derived from a different source or species.

[0049] As used herein, the term "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] As used herein, the term "conditionally active antibody" refers to an antibody that is more active under the conditions of the tumor microenvironment compared to the conditions of the non-tumor microenvironment. Conditions in the tumor microenvironment include a lower pH, higher concentrations of lactic acid and pyruvic acid, hypoxia, lower concentrations of glucose, and a slightly higher temperature compared to the non-tumor microenvironment. For example, a conditionally active antibody is virtually inactive at normal body temperature but is active at a higher temperature in the tumor microenvironment. In yet another aspect, a conditionally active antibody has low activity in normal oxygenated blood but is more active under the hypoxic conditions present in the tumor. In yet another aspect, a conditionally active antibody has low activity at normal physiological pH 7.2 - 7.8 but is more active under the acidic pH 5.8 - 7.0, or 6.0 - 6.8, present in the tumor microenvironment. There are other conditions known to those of skill in the art and which can also be used as conditions in the present invention under which an anti-CTLA4 antibody has different binding affinities for CTLA4.

[0051] As used herein, the term "constitutive" refers to a sustained signaling activity of a receptor kinase that is independent of the presence of a ligand or other activating molecule, as applied, for example, to the activity of CTLA4. Depending on the nature of the receptor kinase, all activity may be constitutive, or the activity of the receptor may be further activated by the binding of other molecules (e.g., ligands). Cellular events that result in the activation of receptor kinases are well known to those of skill in the art. For example, activation may include oligomerization into higher order receptor complexes, such as dimerization, trimerization, etc. The complex may include a single type of protein, i.e., a homomeric complex. Alternatively, the complex may include at least two different types of proteins, i.e., a heteromeric complex. Complex formation can be caused, for example, by overexpression of the normal or mutant form of the receptor on the surface of the cell. Complex formation can also be caused by specific mutations or mutations in the receptor.

[0052] As used herein, the term "cytostatic agent" refers to a compound or composition that halts cell growth either in vitro or in vivo. Thus, a cytostatic agent may significantly reduce the proportion of cells in the S phase. Further examples of cytostatic agents include agents that block cell cycle progression by inducing a G0 / G1 arrest or an M phase arrest. Trastuzumab (HERCEPTIN®), a humanized anti-Her2 antibody, is an example of a cytostatic agent that induces a G0 / G1 arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Certain agents that arrest G1, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and DNA alkylating agents such as ara-C, also spill over into an S phase arrest. Additional information can be found in Mendelsohn and Israel, eds., "The Molecular Basis of Cancer", Section 1, the article by Murakami et al. entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (W.B. Saunders, Philadelphia, 1995), for example on page 13. Taxanes (paclitaxel and docetaxel) are both anti-cancer agents derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the construction of microtubules from tubulin dimers, stabilize the microtubules by preventing depolymerization, and result in the inhibition of mitosis within the cell.

[0053] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 I 131 I 125 Y 90 Re 186 Re 188 Sm 153 Bi 212 P 32 Pb 212 and radioisotopes of Lu), chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents), growth inhibitors, enzymes such as nuclease and fragments thereof, antibiotics, toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and various antitumor or anticancer agents disclosed below.

[0054] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (V H -V L ) linked to a light chain variable domain (V L ) of the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains pair with the complementary domains on the other chain and are forced to generate two antigen-binding sites.

[0055] As used herein, the term "detectably label" refers to any substance that can be detected or measured, either directly or indirectly, by physical or chemical means, the detection or measurement of which indicates the presence of CTCs in a sample. Representative examples of useful detectable labels include, but are not limited to: molecules or ions that are directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are detectable by their radioactive properties; molecules or ions that are detectable by their nuclear magnetic resonance or paramagnetic properties. For example, among the groups of molecules that are indirectly detectable based on light absorbance or fluorescence, there are various enzymes that convert an appropriate substrate, for example, from a non-light-absorbing molecule to a light-absorbing molecule, or from a non-fluorescent molecule to a fluorescent molecule.

[0056] As used herein, the term "diagnosis" refers to the determination of a subject's susceptibility to a disease or disorder, the determination of whether a subject currently has a disease or disorder, the prognosis of a subject having a disease or disorder (e.g., identification of pre-metastatic or metastatic cancer states, cancer stage, or responsiveness of cancer to treatment), and the treatment method (e.g., monitoring the subject's condition to provide information regarding the effect or efficacy of treatment). In some embodiments, the diagnostic methods of the present invention are particularly useful in the detection of early-stage cancer.

[0057] As used herein, the term "diagnostic agent" refers to a molecule that can be detected directly or indirectly and is used for diagnostic purposes. The diagnostic agent may be administered to a subject or sample. The diagnostic agent may be provided by itself or conjugated to a vehicle such as a conditionally active antibody.

[0058] As used herein, the term "effector function" refers to the biological activities of antibodies that are attributable to the Fc regions of the antibodies and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0059] As used herein, the term "effective amount" of a drug, e.g., a pharmaceutical formulation, refers to an amount effective for the dosage and period required to achieve the desired therapeutic or prophylactic result.

[0060] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the native sequence and variants thereof. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0061] As used herein, the term "framework" or "FR" refers to the residues of the variable domain other than the residues of the hypervariable regions (HVR or H1 - 3 in the heavy chain, L1 - 3 in the light chain). The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR sequences and FR sequences generally appear in the following order in V H (or V L ): FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4.

[0062] The terms "full - length antibody", "intact antibody", or "whole antibody" refer to an antigen - binding variable region (V H or V L) and refers to an antibody that includes a light chain constant domain (CL) and heavy chain constant domains (CH1, CH2, and CH3). The constant domains can be the constant domains of the native sequence (e.g., the constant domains of the human native sequence) or amino acid sequence variants thereof. Depending on the amino acid sequences of their heavy chain constant domains, full-length antibodies can be assigned to different "classes". There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0063] As used herein, the terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell (including progeny of such cell) into which an exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells" and include primary transformed cells and their progeny regardless of the number of passages. The progeny may not have a nucleic acid content identical to that of the parental cell and may include mutations. Mutant progeny having the same function or biological activity as that selected or screened in the originally transformed cell are included herein.

[0064] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human source that utilizes the repertoire of human antibodies or the coding sequences of other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0065] As used herein, "human consensus framework" refers to human immunoglobulin V L or V HIn the selection of framework sequences, it is the framework that represents the most commonly occurring amino acid residues. Generally, human immunoglobulin V L or V H sequences are selected from subgroups of variable domain sequences. Generally, the subgroups of sequences are subgroups such as those in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for V L , the subgroup is subgroup κI as in Kabat et al. (supra). In one embodiment, for V H , the subgroup is subgroup III as in Kabat et al. (supra).

[0066] As used herein, the term "humanized" antibody refers to a chimeric antibody that includes amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody includes substantially all of at least one, typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally include at least a portion of the constant region of an antibody derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to the antibody that has been humanized.

[0067] As used herein, the term "hypervariable region" or "HVR" refers to each region of the variable domain of an antibody that is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Generally, a native four-chain antibody includes six HVRs, three in V H (H1, H2, H3) and three in V L(L1, L2, L3) There are three of them. HVR generally contains amino acid residues from hypervariable loops and / or "complementary determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901 - 917 1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24 - 34 of L1, 50 - 56 of L2, 89 - 97 of L3, 31 - 35B of H1, 50 - 65 of H2, and 95 - 102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). V H Except for CDR1 of V, CDRs generally contain the amino acid residues that form the hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs", which are the residues that contact the antigen. SDRs are contained within the region of the CDR and are called abbreviated-CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31 - 34 of L1, 50 - 55 of L2, 89 - 96 of L3, 31 - 35B of H1, 50 - 58 of H2, and 95 - 102 of H3 (see Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619 - 1633, 2008). Unless otherwise indicated, HVR residues and other residues of the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra).

[0068] As used herein, the term "immunoconjugate" refers to an antibody or antibody fragment conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent, a chemotherapeutic agent, a radioisotope, or a cell division inhibitor.

[0069] As used herein, the terms "individual" or "subject" refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0070] As used herein, the term "inhibition of cell growth or proliferation" means reducing the growth or proliferation of cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0071] As used herein, the term "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase high performance liquid chromatography (HPLC)). For an overview of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0072] As used herein, the term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid typically contains nucleic acid molecules that are contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0073] As used herein, the term "isolated nucleic acid encoding an anti-CTLA4 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations within a host cell.

[0074] As used herein, the term "ligand-independent" refers to signaling activity that is independent of the presence of a ligand, for example when applied to receptor signaling activity. A receptor having ligand-independent kinase activity does not necessarily prevent binding of a ligand to the receptor and results in further activation of kinase activity.

[0075] As used herein, the term "metastasis" refers to the entire CTLA4-involved process by which cancer cells disseminate from a primary tumor, penetrate lymphatic vessels and / or blood vessels, circulate through the bloodstream, and grow in distal foci (metastases) in normal tissues at other sites in the body. In particular, it refers to cellular events of tumor cells such as proliferation, migration, anchorage independence, avoidance of apoptosis, or secretion of angiogenic factors, which are stimulated or mediated by non-catalytic or catalytic activity of CTLA4 (preferably including CTLA4 phosphorylation and / or CTLA4-mediated signaling) and underlie metastasis.

[0076] As used herein, the term "microenvironment" means any part or region of a tissue or body that has invariant or transient, physical or chemical differences from the tissue or other regions of the body. In the case of a tumor, the term "tumor microenvironment" as used herein refers to the environment in which the tumor exists, including the acellular regions within the tumor and regions immediately outside the tumor tissue but not related to the intracellular compartments of the cancer cells themselves. The tumor and the tumor microenvironment are closely related and constantly interact. The tumor can change its microenvironment, and the microenvironment can affect the growth and spread of the tumor. Typically, the tumor microenvironment has a low pH, within the range of 5.0 to 7.0, or within the range of 5.0 to 6.8, or within the range of 5.8 to 6.8, or within the range of 6.2 to 6.8. On the other hand, normal physiological pH is in the range of 7.2 to 7.8. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients but higher concentrations of lactate compared to plasma. Additionally, the tumor microenvironment can have a temperature that is 0.3 to 1 °C higher than normal physiological temperature. The tumor microenvironment is discussed in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, which is hereby incorporated by reference in its entirety. The term "non-tumor microenvironment" refers to the microenvironment at sites other than tumors.

[0077] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for, e.g., naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies specific for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is specific for a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used according to the present invention can be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals that include all or part of the human immunoglobulin loci, and such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0078] As used herein, the term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. A naked antibody may be present in a pharmaceutical formulation.

[0079] As used herein, the term "natural antibody" refers to naturally occurring immunoglobulin molecules having a variety of structures. For example, natural IgG antibodies are approximately 150,000 Dalton heterotetrameric glycoproteins composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (V H ), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL )), and subsequently has a constant light chain (C L ) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0080] As used herein, the term "package insert" refers to the instructions customarily included in the commercial package of a therapeutic product and includes information about indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0081] As used herein, the term "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide sequence, after aligning the sequences, introducing gaps as necessary to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the percent value of amino acid sequence identity is generated using the ALIGN-2 computer program for sequence comparison. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office (Washington D.C., 20559) together with a user documentation and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program needs to be compiled for use on a UNIX operating system including Digital UNIX V4.0D. All sequence comparison parameters are set by and do not vary with the ALIGN-2 program.

[0082] In the situation where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A with, to, or against a given amino acid sequence B (or it can also be expressed as a given amino acid sequence A having or containing a specific amino acid sequence identity % with, to, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y Where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. When the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be understood that the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used in this specification are obtained as described in the previous paragraph using the ALIGN-2 computer program.

[0083] As used herein, the term "pharmaceutical preparation" refers to a preparation in a form that enables the biological activity of the active ingredient contained therein and does not contain additional ingredients that are toxic and unacceptable to the subject to which the preparation is administered.

[0084] As used herein, the term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0085] As used herein, the terms "purified" and "isolated" refer to an antibody or nucleotide sequence according to the present invention, and indicate that the indicated molecule is present in the substantial absence of other biopolymers of the same type. As used herein, the term "purified" preferably means that at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of biopolymers of the same type are present. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide, although the molecule may contain some additional bases or moieties that do not have a deleterious effect on the basic characteristics of the composition.

[0086] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric antibody, a humanized antibody or a human antibody, or an antigen-binding fragment thereof) expressed by a recombinant host cell containing a nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 cells and NS0 cells), baby hamster kidney (BHK), Hela cells and Vero cells, (2) insect cells, such as sf9, sf21 and Tn5, (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or the genus Aspergillus (e.g., Aspergillus niger), (5) bacterial cells, such as Escherichia.coli cells or Bacillus subtilis cells, etc.

[0087] As used herein, the term "CTLA4" has the amino acid sequences described in U.S. Patent Nos. 5,434,131, 5,844,095, and 5,851,795, or any portion or derivative thereof, and refers to an immune checkpoint that binds to and recognizes B7 or interferes with B7 so as to block binding to CD28 and / or CTLA4 (e.g., endogenous CD28 and / or CTLA4). In certain embodiments, the extracellular domain of wild-type CTLA4 begins with methionine at position +1 and ends with aspartic acid at position +124, or the extracellular domain of wild-type CTLA4 begins with alanine at position -1 and ends with aspartic acid at position +124. Wild-type CTLA4 is a cell surface protein having an N-terminal extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic domain. The extracellular domain binds to target molecules, such as B7 molecules. In cells, the naturally occurring wild-type CTLA4 protein is translated as an immature polypeptide that includes a signal peptide at the N-terminus. The immature polypeptide undergoes post-translational processing, including cleavage and removal of the signal peptide, to produce a CTLA4 cleavage product having a newly generated N-terminus that is different from the N-terminus of the immature form. One of ordinary skill in the art will understand that additional post-translational processing may occur to remove one or more amino acids from the newly generated N-terminus of the CTLA4 cleavage product. Alternatively, the signal peptide may not be completely removed, resulting in a molecule that begins before the common start amino acid methionine. Thus, the mature CTLA4 protein may begin with methionine at position +1 or alanine at position -1. The mature form of the CTLA4 molecule includes the extracellular domain or any portion thereof.

[0088] The term "therapeutically effective amount" of the antibodies of the present invention means an amount of antibody sufficient to treat the cancer with a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the antibodies and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific antibody being used, the specific composition being used, the patient's age, weight, general health, sex, and diet, the time of administration, route of administration, and excretion rate of the specific antibody being used, the duration of the treatment, drugs used in combination with or simultaneously with the specific antibody being used, as well as similar factors known in the medical arts. For example, it is known in the art to initiate administration of a compound at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0089] As used herein, the term "single-chain Fv" ("scFv") refers to a covalently linked V H ::V L heterodimer, which is typically expressed from a gene fusion encoding a V H and a V L linked by a peptide-encoded linker. "dsFv" is a V H ::V L heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments can be spontaneously formed by the association of monovalent scFvs or generated by linking monovalent scFvs with a peptide linker (such as bivalent sc(Fv)2).

[0090] The terms "treatment", "treating", or "treatment" as used herein, refer to a clinical intervention in an attempt to alter the natural course of an individual being treated, which can be performed for prophylaxis or in any stage of a clinical pathological process. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequence of a disease, prevention of metastasis, reduction of the rate of disease progression, remission or palliation of a disease state, and improvement of remission or prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or to slow the progression of a disease.

[0091] As used herein, the term "tumor" refers to all tumor cell growth and proliferation, and all precancerous and cancerous cells and tissues, whether malignant or benign. The terms "cancer", "cancerous", "proliferative disorder", "growth disorder", and "tumor" are not mutually exclusive as referred to herein.

[0092] As used herein, the term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. Further, V H or V L domains from an antibody that binds an antigen can be used to isolate antibodies that bind a particular antigen, each with a complementary V L or V HA library of domains can be screened. See, for example, Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993, Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.

[0093] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they have been introduced. Certain vectors can induce the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0094] For illustrative purposes, the principles of the present invention are described by reference to various illustrative embodiments. While specific embodiments of the present invention are specifically described herein, those skilled in the art will readily understand that the same principles are equally applicable to and can be used in other systems and methods. Before describing the disclosed embodiments of the present invention in detail, it is to be understood that the present invention is not limited in its application to the details of any particular embodiment shown. In addition, the terminology used herein is for the purpose of description and not of limitation. Further, a particular method is described with reference to steps presented herein in a particular order, but in many cases these steps can be performed in any order as will be understood by those skilled in the art, and thus the novel method is not limited to the particular arrangement of steps disclosed herein.

[0095] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Further, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. The terms "comprising", "including", "having", and "constructed from" may also be used interchangeably.

[0096] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percentages, ratios, reaction conditions, etc., used in this specification and the claims are to be understood as being modified in all instances by the term "about" whether or not the term "about" is present. Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and the claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported significant digits and in reference to the normal rounding procedure. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0097] It is to be understood that each component, compound, substituent, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each of the other components, compounds, substituents, or parameters disclosed herein.

[0098] In addition, each amount / value or range of amounts / values of each component, compound, substituent, or parameter disclosed in this specification should be construed as being disclosed in combination with each amount / value or range of amounts / values of any other component(s), compound(s), substituent, or parameter(s) disclosed in this specification. Thus, for the purposes of this description, it should be understood that any combination of amounts / values or ranges of amounts / values for two or more components(s), compound(s), substituent(s), or parameters disclosed in this specification is also disclosed in combination with each other.

[0099] It is further understood that each lower limit of each range disclosed in this specification should be construed as being disclosed in combination with each upper limit of each range disclosed in this specification for the same component, compound, substituent, or parameter. Thus, the disclosure of two ranges should be construed as the disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. The disclosure of three ranges should be construed as the disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the description or an example should be construed as the disclosure of either a lower limit or an upper limit of a range, and thus can form a range of that component, compound, substituent, or parameter in combination with any other lower limit or upper limit or specific amount / value of the same component, compound, substituent, or parameter disclosed elsewhere in this application.

[0100] A. Anti-CTLA4 antibody In one aspect, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to human CTLA4 protein. The isolated heavy chain variable region polypeptide comprises three complementarity determining regions having H1, H2, and H3, The H1 sequence is GFTFSHYTMH (SEQ ID NO: 1), The H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (SEQ ID NO: 2), The H3 sequence is TGWLGPFDX8 (SEQ ID NO: 3), wherein X1 is S or D, X2 is D, H or I, X3 is N or Y, X4 is Y or I, X5 is Y or E, X6 is D or K, X7 is V or M, and X8 is Y or I.

[0101] Figure 1 shows an alignment of exemplary isolated heavy chain variable regions of the invention, with complementarity determining regions H1, H2, and H3 boxed.

[0102] In another aspect, the invention provides an isolated light chain variable region polypeptide that specifically binds to a human CTLA4 protein. The isolated light chain variable region polypeptide comprises three complementarity determining regions having sequences L1, L2, and L3, The L1 sequence is RX9SQX 10 X 11 GSSYLA (SEQ ID NO: 4), The L2 sequence is GAFSRATGX 12 (SEQ ID NO: 5), The L3 sequence is QQDGSSPWT (SEQ ID NO: 6), wherein X9 is A or I, X 10 is Y, S or H, X 11 is V or G, X 12 is V or I.

[0103] Figure 2 shows an alignment of exemplary isolated light chain variable regions of the invention, with complementarity determining regions L1, L2, and L3 boxed.

[0104] The isolated heavy chain variable region polypeptide and the isolated light chain variable region polypeptide of the invention were each obtained from a parent antibody using the method disclosed in U.S. Patent No. 8,709,755. Methods for generating the isolated heavy chain variable region polypeptide and the isolated light chain variable region polypeptide, as well as methods for generating antibodies and antibody fragments, are disclosed in U.S. Patent No. 8,709,755, which is hereby incorporated herein by reference.

[0105] In another aspect, the present invention includes the heavy chain variable regions shown in FIG. 1 and the light chain variable regions shown in FIG. 2. The amino acid sequences of the 16 heavy chain variable regions in FIG. 1 are shown in SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. The amino acid sequences of the 16 light chain variable regions in FIG. 1 are shown in SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37. Antibodies and antibody fragments comprising these heavy chain variable regions and light chain variable regions can specifically bind to human CTLA4. Antibodies or antibody fragments comprising a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have a higher binding affinity for CTLA4 at the pH of the tumor microenvironment (e.g., pH 6.0 - 6.2) than at the pH of the non-tumor microenvironment (e.g., pH 7.4). As a result, the anti-CTLA4 antibodies or antibody fragments have a higher binding affinity for CTLA4 in the tumor microenvironment compared to their binding affinity for CTLA4 in the normal tissue microenvironment.

[0106] Accordingly, the anti-CTLA4 antibodies or antibody fragments of the present invention have reduced side effects compared to monoclonal anti-CTLA4 antibodies known in the art due to their reduced binding affinity for CTLA4 in the normal tissue microenvironment, and have equivalent efficacy. These features enable the delivery of higher doses of these anti-CTLA4 antibodies or antibody fragments to patients, and thus provide a more effective treatment option.

[0107] The present invention includes those having a heavy chain variable region and a light chain variable region shown in FIGS. 1 to 2, and amino acid sequences of SEQ ID NOs: 7 to 38, and the present invention also includes variants thereof that can specifically bind to human CTLA4. In some embodiments, these variants have different H2, H3, I1 and I2 sequences. In other embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region outside the complementarity determining regions can be mutated according to the principles of substitution, insertion and deletion discussed in the present application. In still further embodiments, the constant region can be modified to provide variants.

[0108] When deriving these variants, the processes described herein serve as a guide. Variants of the heavy chain variable region and the light chain variable region can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy chain variable region and the light chain variable region, or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequences of the heavy chain variable region and the light chain variable region, and / or insertions into residues, and / or substitutions of residues. Any combination of deletions, insertions, and substitutions can be made to arrive at the antibodies or antibody fragments of the present invention, provided that they have the desired characteristics, such as antigen binding to human CTLA4 and / or conditional activity.

[0109] Substitution, insertion, and deletion variants In certain embodiments, antibody or antibody fragment variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include CDRs and framework regions (FRs). Conservative substitutions are shown in Table 1 under the heading "Conservative substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary substitutions" and are further described below with respect to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody or antibody fragment of interest, and the product may be screened for the desired activity, such as retention / improvement of antigen binding or reduction of immunogenicity.

Table 1

[0110] Amino acids can be grouped according to the properties of their common side chains: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues affecting chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0111] Non-conservative substitutions involve exchanging one member of one of these classes for another.

[0112] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., improvement) in a particular biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have a particular biological property of the parent antibody that is substantially retained. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, the variant antibodies are displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0113] For example, in order to improve antibody affinity, modifications (e.g., substitutions) may be made in the CDRs. Such modifications can be made in CDR “hot spots,” i.e., residues encoded by codons that mutate frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in the SDR (a-CDR), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and then reselecting is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide site-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants having the desired affinity. Another method for introducing diversity involves a CDR-specific approach in which some CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0114] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody or antibody fragment to bind antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in the CDRs. Such modifications can be outside of the CDR “hot spots” or in the SDR. The variant V H and V L In certain embodiments of the sequences, each CDR is either unmodified or contains no amino acid substitutions, one amino acid substitution, two amino acid substitutions, or three or more amino acid substitutions.

[0115] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989. In this method, target residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody or antibody fragment and the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the first substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex identifies the contact points between the antibody or antibody fragment and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or may be excluded. Variants may be screened to determine whether they contain the desired properties.

[0116] Insertions of amino acid sequences include amino-terminal fusions and / or carboxyl-terminal fusions (chain lengths ranging from 1 residue to polypeptides of 100 residues or more), as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibodies include fusions of an enzyme (e.g., ADEPT) or polypeptide that increase the serum half-life of the antibody at the N-terminal or C-terminal of the antibody.

[0117] Modifications of the amino acid sequences of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Only the CDRs in the V H and V L of antibodies derived from non-human animals are simply replaced with the V H and V LWhen a humanized antibody is produced by transplantation into the FR in, the antigen-binding activity is known to decrease as compared with the antigen-binding activity of the original antibody derived from a non-human animal. Not only in the CDR but also in the FR, some amino acid residues of the non-human antibody V H and V L are thought to be directly or indirectly related to the antigen-binding activity. Therefore, substituting these amino acid residues with different amino acid residues derived from the FR of human antibody V H and V L will likely reduce the binding activity. To solve this problem, in an antibody transplanted with human CDR, among the amino acid sequences of the FR of human antibody V H and V L , it is necessary to attempt to identify amino acid residues that are directly related to the binding of the antibody, or amino acid residues that interact with the amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to the binding to the antigen. The decreased antigen-binding activity can be increased by substituting the identified amino acids with the amino acid residues of the original antibody derived from a non-human animal.

[0118] Modifications and changes are made to the structure of the antibodies of the present invention and the DNA sequences encoding them, and functional molecules encoding antibodies that still have the desired characteristics can be obtained.

[0119] When making changes to the amino acid sequence, the hydrophilicity index of the amino acids may be considered. The importance of the hydrophilic amino acid index in imparting biological functions of interaction to proteins is generally understood in the art. The relative hydrophilic characteristics of amino acids are accepted to contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophilicity index based on their hydrophobic and charge characteristics, which are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0120] Furthermore, a further object of the present invention also includes functionally conserved variants of the antibodies of the present invention.

[0121] A "functionally conserved variant" is one in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, and includes, but is not limited to, amino acid substitutions with amino acids having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those shown as conserved amino acids may be different in the protein, and as a result, the percent sequence similarity of the proteins or amino acids between any two proteins with similar functions may vary, e.g., when similarity is determined according to an alignment scheme such as a clustering method based on the MEGALIGN algorithm, it can be between 70% and 99%. A "functionally conserved variant" also includes polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% when determined by the BLAST or FASTA algorithm, and having the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0122] Two amino acid sequences are "substantially homologous" or "substantially similar" if more than 80%, preferably more than 85%, preferably more than 90% of the amino acids are identical, or if more than about 90%, preferably more than 95% are similar (functionally identical) over the entire length of the shorter sequence. Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the sequence comparison algorithms such as BLAST, FASTA.

[0123] For example, certain amino acids can be substituted by other amino acids in the protein structure without significantly impairing their activity. The ability and properties of protein interactions define the biological functional activity of the protein. Therefore, within the protein sequence, and of course within the sequence encoded by its DNA, certain amino acid substitutions can be made, yet proteins with similar properties can still be obtained. Thus, it is contemplated that various modifications can be made to the sequence of the antibody or antibody fragment of the present invention, or the corresponding DNA sequence encoding the antibody or antibody fragment, without significantly impairing their biological activity.

[0124] In the art, it is known that certain amino acids may be substituted by other amino acids having similar hydrophilicity indices or scores, still resulting in proteins having similar biological activity, i.e., still obtaining proteins equivalent in biological function.

[0125] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the substituents of the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various features described above are known to those skilled in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, and valine, leucine and isoleucine.

[0126] Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can conveniently be achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0127] When the antibody contains an Fc region, the carbohydrates that bind thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, generally attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants with certain improved properties.

[0128] In one embodiment, an antibody variant is provided that has a carbohydrate structure lacking fucose that binds (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, as described in, for example, WO2008 / 077546. Asn297 refers to the asparagine residue located at position approximately 297 within the Fc region (Eu numbering of Fc region residues), but Asn297 can be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol., vol. 336, pp. 1239-1249, 2004, Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004.Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986, US Patent Publication No. US2003 / 0157108A, and WO2004 / 056312A1 (especially Example 11)), and knockout cell lines such as α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006, and WO2003 / 085107).

[0129] Antibody variants containing a bisected oligosaccharide are further provided. For example, the bisected oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878, US Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0130] Fc region variant In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants can include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0131] In certain embodiments, the invention contemplates antibody variants that have some, but not all, effector functions, which would be desirable candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest include U.S. Patent No. 5,500,362 (see also, e.g., Hellstrom et al. Proc. Nat’l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986), and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985, U.S. Patent No. 5,821,337 (see also Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model as disclosed, for example, in Clynes et al. Proc. Nat’l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp. 163-171, 1996, Cragg, M. S. et al., Blood, vol. 101, pp. 1045-1052, 2003, and Cragg, M. S, and M. J. Glennie, Blood, vol. 103, pp. 2738-2743, 2004). Also, FcRn binding and in vivo clearance / half-life determination can be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int’l. Immunol., vol. 18, pp. 1759-1769, 2006).

[0132] Antibodies having reduced effector function include antibodies having one or more substitutions of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc variant having substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0133] Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

[0134] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0135] In some embodiments, the Fc region is modified to result in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC). See, e.g., U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0136] Antibodies with improved binding to the neonatal Fc receptor (FcRn) (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994) involved in the transfer of maternal IgG to the fetus and increased half-life are described in US2005 / 0014934. These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region (e.g., substitution of residue 434 of the Fc region, U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988, U.S. Patent Nos. 5,648,260, 5,624,821, and WO94 / 29351.

[0137] Cysteine-modified antibody variant In certain embodiments, it may be desirable to generate a cysteine-modified antibody, e.g., a "thioMAb", in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residue substitution occurs at an accessible site of the antibody. By replacing these residues with cysteine, a reactive thiol group is placed at an accessible site of the antibody and, as further described herein, the antibody can be conjugated to another moiety such as a drug moiety or a linker-drug moiety to generate an immunoconjugate. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and 5400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0138] Antibody derivative In certain embodiments, the antibodies or antibody fragments provided herein can be further modified to include additional non - proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing the antibody or antibody fragment include, but are not limited to, water - soluble polymers. Non - limiting examples of water - soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly - 1,3 - dioxolane, poly - 1,3,6 - trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n - vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be a polymer of any molecular weight and can be a branched or unbranched polymer. The number of polymers attached to the antibody or antibody fragment may vary, and when two or more polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody or antibody fragment to be improved, whether the derivative is to be used in therapy under a given set of conditions, etc.

[0139] In another embodiment, a conjugate of an antibody or antibody fragment that can be selectively heated by exposure to radiation and a non-proteinaceous moiety is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation can be of any wavelength and includes wavelengths that do not damage normal cells but heat the non-proteinaceous moiety to a temperature at which cells in proximity to the antibody-non-proteinaceous moiety die.

[0140] In another aspect, the invention provides an anti-CTLA4 antibody or antibody fragment comprising an isolated heavy chain variable region polypeptide or an isolated light chain variable region polypeptide. The isolated heavy chain variable region polypeptide comprises H1, H2, and H3 regions having SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. The isolated light chain variable region polypeptide comprises L1, L2, and L3 regions having SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37.

[0141] The anti-CTLA4 antibody or antibody fragment of the invention has a higher binding affinity for CTLA4 under conditions in the tumor microenvironment than under conditions in the non-tumor microenvironment. In one embodiment, the conditions in the tumor microenvironment and the non-tumor microenvironment are both pH. Thus, the anti-CTLA4 antibody or antibody fragment of the invention can selectively bind to CTLA4 at a pH of about 5.0 to 6.8, but has a lower binding affinity for CTLA4 at a pH of about 7.2 to 7.8 encountered in a normal physiological environment. As shown in Examples 2-3, the anti-CTLA4 antibody or antibody fragment has a higher binding affinity for CTLA4 at pH 6.0 than at pH 7.4.

[0142] In certain embodiments, the anti-CTLA4 antibody or antibody fragment of the invention has a dissociation constant (Kd) with CTLA4 of about 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, or 10 -8 M to 10 -13 M, or 10 -9 M to 10 -13 M) under conditions in the tumor microenvironment. In one embodiment, the ratio of the Kd of the antibody or antibody fragment with CTLA4 at a value of the conditions in the tumor microenvironment to the Kd at a different value of the same conditions in the non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0143] In one embodiment, the Kd is measured by a radiolabeled antigen binding assay (RIA) performed on the Fab version of the antibody of interest and its antigen using the following assay. The solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with the minimum concentration of ( 125 I) radiolabeled antigen in the presence of a titration series of unlabeled antigen and then capturing the bound antigen using a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of a capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked for 2-5 hours at room temperature (approximately 23° C.) with 2% (w / v) bovine serum albumin in PBS. In a non-adsorbing plate (Nunc #269620), 100 pM or 26 pM of 125Mix the I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although the incubation may continue for a longer time (e.g., about 65 hours) to ensure reaching equilibrium. Thereafter, transfer the mixture to a capture plate and incubate at room temperature (e.g., for 1 hour). Then remove the solution and wash the plate 8 times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20 (trademark), Packard) and count the plate for 10 minutes with a TOPCOUNT (trademark) gamma counter (Packard). Select the concentration of each Fab that gives 20% or less of the maximum binding for use in a competitive binding assay.

[0144] According to another embodiment, the Kd is measured at about 10 response units (RU) at 25° C. using a surface plasmon resonance assay with a BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, N.J.) and an immobilized antigen CM5 chip. Briefly, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve a coupled protein of about 10 response units (RU). After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serial 2-fold dilutions of the Fab (0.78 nM to 500 nM) are injected at 25° C. at a flow rate of about 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN-20 (trademark)) surfactant (PBST). Association rate (k on) and dissociation rate (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on rate exceeds 10 6 M -1 s -1 in the above surface plasmon resonance assay, the on rate can be determined by increasing the concentration of the antigen as measured by a spectrometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) equipped with a stirred cuvette using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM antigen-antibody (Fab form) in PBS (pH 7.2) at 25°C.

[0145] The anti-CTLA4 antibody of the present invention can be a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, anti-CTLA4 antibody fragments such as Fv, Fab, Fab’, Fab’-SH, scFv, diabody, triabody, tetrabody, or F(ab’)2 fragments formed from antibody fragments, and bispecific antibodies are used. In another embodiment, the antibody is a full-length antibody such as an intact IgG antibody, or another antibody class or isotype defined herein. For a review of specific antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as WO93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab’)2 fragments that contain salvage receptor binding epitope residues and have an increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0146] The diabody of the present invention can be bivalent or bispecific. For examples of diabodies, see EP404,097, WO1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993. Also, examples of triabodies and tetra-bodies are described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003.

[0147] In some embodiments, the invention includes single domain antibody fragments and includes all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (see, e.g., Domantis, Inc., Waltham, Mass., and U.S. Patent No. 6,248,516 B1).

[0148] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells (e.g., E. coli or phage) described herein.

[0149] In some embodiments, the anti-CTLA4 antibody of the invention can be a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class switched" antibody in which the class or subclass of the antibody is changed compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0150] In certain embodiments, the chimeric antibodies of the invention are humanized antibodies. Typically, such non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody contains one or more variable domains, with the CDR (or a portion thereof) being derived from a non-human antibody and the FR (or a portion thereof) being derived from a human antibody sequence. Additionally, a humanized antibody may optionally contain at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0151] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further described, for example, in Riechmann et al., Nature, vol. 332, pp. 323-329, 1988, Queen et al., Proc. Nat’l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989, U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR(a-CDR) grafting), Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing “resurfacing”), Dall’Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing “FR shuffling”), and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing the “guided selection” approach to FR shuffling).

[0152] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993), framework regions derived from consensus sequences of human antibodies of certain subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992 and Presta et al. J. Immunol., vol. 151, p. 2623, 1993), human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008), and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996).

[0153] In some embodiments, the anti-CTLA4 antibodies of the invention are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for CTLA4 and the other is for another antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of CTLA4. The bispecific antibody can also be used to localize a cytotoxic agent to cells expressing CTLA4. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment.

[0154] Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983, WO93 / 08829, and Traunecker et al., EMBO J. vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Bispecific antibodies can also be made by manipulating electrostatic steering effects for making antibody Fc-heterodimer molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985), producing bispecific antibodies using leucine zippers (see, for example, Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), using "diabody" technology for making bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993), and using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., vol. 152, pp. 5368-5374, 1994), as well as by preparing trispecific antibodies (described, for example, in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991).

[0155] Also included herein are modified antibodies having three or more functional antigen-binding sites, including "Octopus antibodies" (see, for example, US2006 / 0025576A1).

[0156] The anti-CTLA4 antibodies or antibody fragments of the present invention can be produced using the recombinant methods and compositions described in detail in US2016 / 0017040.

[0157] The physical / chemical properties and / or biological activities of the anti-CTLA4 antibodies or antibody fragments of the present invention can be tested and measured by various assays known in the art. Some of these assays are described in U.S. Patent No. 8,853,369.

[0158] B. Immunoconjugate In another aspect, the present invention also provides an immunoconjugate comprising an anti-CTLA4 antibody or antibody fragment conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope.

[0159] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein an antibody or antibody fragment is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425235B1), auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatin, calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer Res., vol. 53, pp. 3336-3342, 1993, and Lode et al., Cancer Res., vol. 58, pp. 2925-2928, 1998), anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med.Chem., vol. 13, pp. 477-523, 2006, Jeffrey et al., Bioorganic & Med.Chem.Letters, vol. 16, pp. 358-362, 2006, Torgov et al., Bioconj.Chem., vol. 16, pp. 717-721, 2005, Nagy et al., Proc.Natl.Acad.Sci.USA, vol. 97, pp. 829-834, 2000, Dubowchik et al., Bioorg.& Med.Chem.Letters, vol. 12, vol. 1529-1532, 2002, King et al., J.Med.Chem., vol. 45, pp.(See, e.g., U.S. Patent Nos. 4,336,4343, 2002, and U.S. Patent No. 6,630,579), methotrexate, vindesine, taxanes (such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel), trichothecenes, and CC1065.

[0160] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0161] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of radioactive conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioisotopes of Lu. When a radiolabeled conjugate is used for detection, it may include radioatoms for scintigraphy studies, such as tc99m or I123, or iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron, or spin labels for nuclear magnetic resonance (NMR) imaging methods (also known as magnetic resonance imaging, MRI).

[0162] Conjugates of antibodies / antibody fragments and cytotoxic agents can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science, vol. 238, pp. 1098-, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug intracellularly. For example, an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker, or a linker containing a disulfide (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992, U.S. Patent No. 5,208,020) may be used.

[0163] Immunoconjugates herein include, but are not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A.) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), and expressly contemplate conjugates prepared with crosslinking reagents.

[0164] Exemplary embodiments of an ADC include an antibody or antibody fragment (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that conjugates the Ab to the D. In some embodiments, the antibody is conjugated to the linker moiety (L) via one or more amino acid residues such as lysine and / or cysteine.

[0165] An exemplary ADC has the formula I as p (Ab-(L-D)), where p is from 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs of formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 modified cysteine amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine residues are already present in the antibody without using a modification, in which case the existing free cysteine residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to generate one or more free cysteine residues.

[0166] i) Exemplary Linkers The "linker" (L) is bifunctional or polyfunctional and can be used to link one or more moieties, such as a drug moiety (D), to an antibody or antibody fragment (Ab) to form an immunoconjugate such as an ADC of Formula I. In some embodiments, the ADC can be prepared using a linker having a reactive functionality for covalently bonding to the drug and the antibody. For example, in some embodiments, the cysteine thiol of an antibody or antibody fragment (Ab) can form a bond with a reactive functional group of the linker or drug-linker intermediate to generate an ADC.

[0167] In one aspect, the linker has a functionality capable of reacting with a free cysteine present on the antibody to form a covalent bond. Non-limiting exemplary such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, the conjugation method on page 766 of Klussman, et al, Bioconjugate Chemistry, vol. 15, pp. 765-773, 2004.

[0168] In some embodiments, the linker has a functionality capable of reacting with an electrophilic group present on the antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on the antibody to form a covalent bond to the antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide.

[0169] The linker may contain one or more linker components. Exemplary linker components include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), and 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (MCC). Various linker components are known in the art, and some of them are described below.

[0170] The linker can be a "cleavable linker" that promotes drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., those containing hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photo-labile linkers, or linkers containing disulfide (Cancer Research, vol. 52, pp. 127-131, 1992, U.S. Patent No. 5,208,020).

[0171] In certain embodiments, the linker is -A a -W w -Y y - has the following formula II, wherein A is an "extension unit", a is an integer from 0 to 1, W is an "amino acid unit", w is an integer from 0 to 12, Y is a "spacer unit", and y is 0, 1, or 2. An ADC comprising the linker of formula II has the formula I(A): Ab-(A a -W w -Y y -D) p wherein Ab, D, and p are defined as above for formula I. Exemplary embodiments of such linkers are described in U.S. Patent No. 7,498,298.

[0172] In some embodiments, the linker component includes an "extension unit" (A) that links the antibody to another linker component or drug moiety. Non-limiting, exemplary extension units are shown below (the wavy lines indicate the sites of covalent attachment to the antibody, drug, or additional linker component).

Chemical Formula

[0173] In some embodiments, the linker component includes an "amino acid unit" (W). In some such embodiments, the amino acid unit enables cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases (e.g., lysosomal enzymes) (Doronina et al., Nat. Biotechnol., vol. 21, pp. 778 - 784, 2003). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit can include naturally occurring amino acid residues and / or minor amino acids and / or non-natural amino acid analogs, such as citrulline amino acid units that can be designed and optimized for enzymatic cleavage by specific enzymes, e.g., tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0174] Typically, a peptide linker can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods (e.g., E. Schroder and K. Lubke (1965) “The Peptides”, volume 1, pp 76 - 136, Academic Press).

[0175] In some embodiments, the linker component includes a “spacer unit” (Y) that directly or through an extension unit and / or amino acid unit couples an antibody to a drug moiety. The spacer unit can be “self - immolative” or “non - self - immolative”. A “non - self - immolative” spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety upon cleavage of the ADC. Examples of non - self - immolative spacer units include, but are not limited to, glycine spacer units and glycine - glycine spacer units. In some embodiments, enzymatic cleavage of an ADC containing a glycine - glycine spacer unit by a tumor cell - associated protease results in the release of the glycine - glycine - drug moiety from the remainder of the ADC. In some such embodiments, the glycine - glycine - drug moiety is subjected to a hydrolysis step in the tumor cell, and thus the glycine - glycine spacer unit is cleaved from the drug moiety.

[0176] The "self-destructive" spacer unit enables the release of the drug moiety. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is linked to an amino acid unit via an amide bond to form a carbamate, methylcarbamate, or carbonate between the benzyl alcohol and the drug (Hamann et al. Expert Opin. Ther. Patents, vol. 15, pp. 1087-1103, 2005). In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-destructive linker has the following structure, [Chemical formula] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, m is an integer in the range of 0 to 4, X may be one or more additional spacer units or absent, and p ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Non-limiting and exemplary X spacer units are [Chemical formula] wherein R1 and R2 are independently selected from H and C1-C6 alkyl. In some embodiments, R1 and R2 are each -CH3.

[0177] Other examples of self - cleaving spacers include, but are not limited to, aromatic compounds electronically similar to the PAB group, such as 2 - aminoimidazole - 5 - methanol derivatives (U.S. Patent No. 7,375,078, Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237 -, 1999), and ortho or para aminobenzyl acetals. In some embodiments, substituted and unsubstituted 4 - aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol. 2, pp. 223 -, 1995), appropriately substituted bicyclo[2.2.1] ring systems and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., vol. 94, p. 5815 -, 1972), and spacers that undergo cyclization upon hydrolysis of the amide bond, such as 2 - aminophenylpropionic acid amide (Amsberry et al, J. Org. Chem., vol. 55, p. 5867, 1990) can be used. The attachment of a drug to the α - carbon of a glycine residue is another example of a self - cleaving spacer that can be useful in an ADC (Kingsbury et al., J. Med. Chem., vol. 27, p. 1447, 1984).

[0178] In some embodiments, the linker L may be a dendritic - type linker for covalently attaching two or more drug moieties to an antibody via a branched polyfunctional linker moiety (Sun et al. Bioorganic & Medicinal Chemistry Letters, vol. 12, pp. 2213 - 2215, 2002, Sun et al., Bioorganic & Medicinal Chemistry, vol. 11, pp. 1761 - 1768, 2003). Dendritic linkers can increase the molar ratio of drug to antibody (i.e., the loading related to the potency of the ADC). Thus, if an antibody has only one reactive cysteine thiol group, multiple drug moieties can be attached via the dendritic linker.

[0179] In the context of an ADC, a non - limiting, exemplary linker is represented by the following formula I: [Chem.] In the formula, R1 and R2 are independently selected from H and C1-C6 alkyl. In some embodiments, R1 and R2 are each -CH3. [Chem.] In the formula, n is from 0 to 12. In some embodiments, n is from 2 to 10. In some embodiments, n is from 4 to 8.

[0180] Further non-limiting and exemplary ADCs include the following structures: [Chem.] Each R is independently H or C1-C6 alkyl, and n is from 1 to 12.

[0181] In some embodiments, the linker is substituted with groups that modulate solubility and / or reactivity. As non-limiting examples, charged substituents such as sulfonate (-SO3 - ) or ammonium can increase the water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent with the antibody and / or drug moiety, depending on the synthetic route used to prepare the ADC, or can facilitate the coupling reaction of D with Ab-L (antibody-linker intermediate) or the coupling reaction of Ab with D-L (drug-linker intermediate). In some embodiments, a portion of the linker is coupled to the antibody and a portion of the linker is coupled to the drug, and then Ab-(linker moiety) a is coupled to drug-(linker moiety) b to form the ADC of formula I.

[0182] The compounds of the present invention are explicitly contemplated to be used to prepare ADCs using, but not limited to, the following linker reagents: bis-maleimide-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyl-oxy) succinimide ester (EMCS), N-[γ-maleimidobutyryloxy] succinimide ester (GMBS), 1,6-hexane-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido) propionate (SBAP), succinimidyl iodoacetate (SIA), succinidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl 6-[(beta-maleimidopropionamido) hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone) benzoate (SVSB), as well as bis-maleimide reagents including: dithiobismaleimide ethane (DTME), 1,4-bismaleimide butane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimide hexane (BMH), bismaleimide ethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below);Bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, the bis-maleimide reagent enables the binding of the thiol group of the cysteine of the antibody to a thiol-containing drug moiety, a linker, or a linker-drug intermediate. Other functional groups that react with the thiol group include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.;

[0183] Certain useful linker reagents can be obtained from various commercial sources such as Pierce Biotechnology, Inc. (Rockford, Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or synthesized according to procedures described in the art (e.g., Toki et al., J. Org. Chem., vol. 67, pp. 1866-1872, 2002, Dubowchik, et al., Tetrahedron Letters, vol. 38, pp. 5257-60, 1997, Walker, J. Org. Chem., vol. 60, pp. 5352-5355, 1995, Frisch et al., Bioconjugate Chem., vol. 7, pp. 180-186, 1995, U.S. Patent No. 6,214,345, WO02 / 088172, US2003 / 130189, US2003 / 096743, WO03 / 026577, WO03 / 043583, and WO04 / 032828).

[0184] Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radio nucleotide to an antibody. See, for example, WO94 / 11026.

[0185] ii) Exemplary drug moieties 1) Maytansines and maytansinoids In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors that act by inhibiting the polymerization of tubulin. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0186] The drug moiety of maytansinoids is an attractive drug moiety in antibody-drug conjugates for the following reasons: (i) it is relatively accessible for preparation by fermentation or chemical modification or derivatization of fermentation products, (ii) it is suitable for derivatization with functional groups suitable for conjugation to an antibody via a non-sulfide linker, (iii) it is stable in plasma, and (iv) it is effective against various tumor cell lines.

[0187] Certain mitansinoid drugs that are suitable for use as mitansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Mitansinoids can also be prepared synthetically according to known methods.

[0188] Exemplary mitansinoid drug moieties include, but are not limited to, those having a modified aromatic ring such as: C-19-dechloro (U.S. Patent No. 4,256,746) (e.g., prepared by lithium aluminum hydride reduction of ansamitocin P2), C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (e.g., prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH), and C-20-demethoxy, C-20-acetyloxy (-OCOR), + / - dechloro (U.S. Patent No. 4,294,757) (e.g., prepared by acylation using acyl chloride), and those having modifications at other positions of the aromatic ring.

[0189] Exemplary mitecinoid drug moieties include those having the following modifications: C-9-SH (U.S. Patent No. 4,424,219) (prepared, for example, by reacting mitolactol with H2S or P2S5), C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Patent No. 4,450,254) (prepared, for example, from Nocardia), C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (prepared, for example, by conversion of mitolactol by Streptomyces), C-15-methoxy (U.S. Patents Nos. 4,313,946 and 4,315,929) (isolated, for example, from Trewia nudiflora), C-18-N-demethyl (U.S. Patents Nos. 4,362,663 and 4,322,348) (prepared, for example, by demethylation of mitolactol by Streptomyces), and 4,5-deoxy (U.S. Patent No. 4,371,533) (prepared, for example, by titanium trichloride / LAH reduction of mitolactol).

[0190] Many positions on the mitecinoid compound are useful as attachment sites. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In some embodiments, the attachment is formed at the C-3 position of mitolactol or a mitolactol analog.

[0191] Mitecinoid drug moieties include those having the following structures:

Chemical formula

[0192] All stereoisomers of the maytansinoid drug moiety are contemplated for the ADCs of the present invention, i.e., any combination of R and S configurations at the chiral carbon (U.S. Patent Nos. 7,276,497, 6,913,748, 6,441,163, 633,410 (RE39151), 5,208,020, Widdison et al (2006) J. Med. Chem. 49:4392-4408). In some embodiments, the maytansinoid drug moiety has the following stereochemistry:

Chemical formula

[0193] Exemplary embodiments of the maytansinoid drug moiety include, but are not limited to, DM1, DM3, and DM4 having the following structures.

Chemical formula

[0194] An exemplary antibody-drug conjugate in which DM1 is linked to the thiol group of the antibody via a BMPEO linker has the following structure and abbreviations:

Chemical formula

[0195] Immunoconjugates containing maytansinoids, methods for making them, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064, US2005 / 0276812A1, and EP0425235B1. See also Liu et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 8618 - 8623, 1996, and Chari et al., Cancer Research, vol. 52, pp. 127 - 131, 1992.

[0196] In some embodiments, the antibody - maytansinoid conjugate can be prepared by chemically conjugating the antibody to the maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020. In some embodiments, an ADC conjugated with an average of 3 - 4 maytansinoid molecules per antibody molecule has shown effectiveness in enhancing the cytotoxicity of target cells without adversely affecting the function or solubility of the antibody. In some cases, even 1 molecule of toxin / antibody is expected to enhance cytotoxicity compared to using a naked antibody.

[0197] Exemplary linkers for making antibody - maytansinoid conjugates include, for example, the linkers described herein and the linkers described in U.S. Patent No. 5,208,020, EP0425235B1, Chari et al., Cancer Research, vol. 52, pp. 127 - 131, 1992, US2005 / 0276812A1, and US2005 / 016993A1.

[0198] (2) Auristatin and dolastatin Drug moieties include dolastatin, auristatin, and their analogs and derivatives (U.S. Patent Nos. 5,635,483, 5,780,588, 5,767,237, and 6,124,431). Auristatin is a derivative of dolastatin-10, a compound from marine mollusks. Without intending to be bound by any particular theory, dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and mitosis and cell division (Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001), as well as anti-cancer (U.S. Patent No. 5,663,149), and anti-fungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The dolastatin / auristatin drug moiety can be conjugated to an antibody via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO02 / 088172, Doronina et al., Nature Biotechnology, vol. 21, pp. 778-784, 2003, Francisco et al., Blood, vol. 102, pp. 1458-1465, 2003).

[0199] Exemplary auristatin embodiments include D, an N-terminal conjugated monomethyl auristatin drug moiety disclosed in U.S. Patent Nos. 7,498,298 and 7,659,241 E and D F are included:

Chemical formula

[0200] In one embodiment, R 3 , R 4 and R 7 are independently isopropyl or sec - butyl, and R 5 is -H or methyl. In an exemplary embodiment, R 3 and R 4 are each isopropyl, R 5 is -H, and R 7 is sec - butyl.

[0201] In yet another embodiment, R 2 and R 6 are each methyl, and R 9 is -H.

[0202] In yet another embodiment, R 8 is, at each occurrence, -OCH3.

[0203] In an exemplary embodiment, R 3 and R 4 are each isopropyl, R 2 and R 6 are each methyl, R 5 is -H, R 7 is sec-butyl, R 8 is, at each occurrence, -OCH3, and R 9 is -H.

[0204] In one embodiment, Z is -O- or -NH-.

[0205] In one embodiment, R 10 is aryl.

[0206] In an exemplary embodiment, R 10 is -phenyl.

[0207] In an exemplary embodiment, when Z is -O-, R 11 is -H, methyl, or t-butyl.

[0208] In one embodiment, when Z is -NH, R 11 is -CH(R 15 )2, wherein R 15 is -(CH2) n -N(R 16 )2 and R 16 is -C1-C8 alkyl or -(CH2) n -COOH.

[0209] In another embodiment, when Z is -NH, R 11 is -CH(R 15 )2 and R 15 is -(CH2) n -SO3H.

[0210] Formula D E An exemplary embodiment of auristatin is MMAE, and the wavy line indicates a covalent bond to the linker (L) of the antibody-drug conjugate: [Chemical formula]

[0211] Formula D E An exemplary embodiment of auristatin is MMAF, and the wavy line indicates a covalent bond to the linker (L) of the antibody-drug conjugate: [Chemical formula]

[0212] Other exemplary embodiments include a monomethylvaline compound having a carboxy modification of phenylalanine at the C-terminus of the pentapeptide auristatin drug moiety (WO2007 / 008848) and a monomethylvaline compound having a modification of the side chain of phenylalanine at the C-terminus of the pentapeptide auristatin drug moiety (WO2007 / 008603).

[0213] Non-limiting and exemplary embodiments of the ADC of Formula I comprising MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF conjugated to an antibody by a cleavable linker that is not proteolytic have been shown to have equivalent activity to immunoconjugates comprising MMAF conjugated to an antibody by a proteolytically cleavable linker (Doronina et al., Bioconjugate Chem., vol. 17, pp. 114 - 124, 2006). In some such embodiments, drug release is thought to be affected by the degradation of the antibody in cells.

[0214] Typically, a peptide-based drug moiety can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid-phase synthesis methods (see, for example, E. Schroder and K. Lubke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press). The auristatin / dolastatin drug moiety can, in some embodiments, be prepared according to the following methods: U.S. Patent Nos. 7,498,298, 5,635,483, 5,780,588, Pettit et al., J. Am. Chem. Soc., vol. 111, pp. 5463-5465, 1998, Pettit et al., Anti-Cancer Drug Design, vol. 13, pp. 243-277, 1998, Pettit et al., Synthesis, vol. 6, pp. 719-725, 1996, Pettit et al., J. Chem. Soc. Perkin Trans. vol. 15, pp. 859-863, 1996, and Doronina, Nat. Biotechnol., vol. 21, pp. 778-784, 2003.

[0215] In some embodiments, a drug moiety of formula D such as MMAE E and a drug moiety of D such as MMAF E as well as drug-linker intermediates and derivatives thereof such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE can be prepared using the methods described in U.S. Patent No. 7,498,298, Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006, and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003 and can then be conjugated to the antibody of interest.

[0216] (3) Calicheamicin In some embodiments, the immunoconjugate comprises an antibody or antibody fragment conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics and their analogs are capable of generating double-stranded DNA breaks at subpicomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; Lode et al., Cancer Research, vol. 58, pp. 2925-2928, 1998). Calicheamicin has an intracellular site of action but, in certain cases, does not readily cross the cell membrane. Thus, in some embodiments, cellular uptake of these agents through antibody-mediated internalization can significantly enhance their cytotoxic effects. Non-limiting and exemplary methods for preparing antibody-drug conjugates having a calicheamicin drug moiety are described, for example, in U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, and 5,767,285.

[0217] (4) Pyrrolobenzodiazepine In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PDB dimer recognizes and binds to a specific DNA sequence. The PBD, a natural product anthramycin, was first reported in 1965 (Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5793-5795, 1965; Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5791-5793, 1965). Subsequently, several PBDs of both natural and analogs have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994), including dimers of the tricyclic PBD skeleton (Chem. Rev. vol. 1994, pp. 433-465 1994; U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). Without intending to be bound by any particular theory, the dimer structure is thought to confer a three-dimensional shape appropriate for isostericity with the minor groove of B-form DNA and fit precisely into the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986). Dimeric PBD compounds having C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al Cancer Res., vol. 70, pp. 6849-6858, 2010; Antonow, J. Med. Chem. vol. 53, pp. 2927-2941, 2010; Howard et al., Bioorganic and Med. Chem. Letters, vol. 19, pp. 6463-6466, 2009).

[0218] The PBD dimer is conjugated to an antibody, and the resulting ADC has been shown to have anti-cancer properties. Non-limiting and exemplary linking sites on the PBD dimer include the 5-membered pyrrolo ring, tethering between PBD units, and the N10-C11 imine group (WO2009 / 016516, US2009 / 304710, US2010 / 047257, US2009 / 036431, US2011 / 0256157, WO2011 / 130598).

[0219] Non-limiting and exemplary PBD dimer components of the ADC are

Chemical formula

[0220] In some embodiments, R and R’ are each independently selected from optionally substituted C 1~12 alkyl, C 3~20 heterocyclic ring, and C 5~20 aryl groups, optionally in relation to the groups NRR’, R, and R’, and together with the nitrogen atom to which they are attached, optionally form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring. In some embodiments, R 9 and R 19 are H. In some embodiments, R 6 and R 16 are H.

[0221] In some embodiments, R 7 and R 17 are both OR 7A where R 7A is optionally substituted C 1~4 alkyl. In some embodiments, R 7Ais Me. In some embodiments, R 7A is Ch2Ph, where Ph is a phenyl group. In some embodiments, X is O. In some embodiments, R 11 is H. In some embodiments, a double bond exists between C2 and C3 in each monomer unit.

[0222] In some embodiments, R 2 and R 12 are independently selected from H and R. In some embodiments, R 2 and R 12 are independently R. In some embodiments, R 2 and R 12 are independently optionally substituted C 5~20 aryl or C 5~7 aryl or C 8~10 aryl. In some embodiments, R 2 and R 12 are independently optionally substituted phenyl, thienyl, naphthyl, pyridyl, quinolinyl, or isoquinolinyl. In some embodiments, R 2 and R 12 are independently selected from =O, =CH2, =CH-R D , and =C(R D )2. In some embodiments, R 2 and R 12 are each =CH2. In some embodiments, R 2 and R 12 are each H. In some embodiments, R 2 and R 12 are each =O. In some embodiments, R 2 and R 12 are each =CF2. In some embodiments, R 2 and / or R 12 are independently =C(R D )2. In some embodiments, R 2 and / or R 12 are independently =CH-R D .

[0223] In some embodiments, R 2 and / or R 12 is =CH-R D when this is the case, each group may independently have any of the structures shown below:

Chemical formula

[0224] The linkers of PBD dimer-val-cit-PAB-Ab and PBD dimer-Phe-Lys-PAB-Ab are cleavable by proteases, while the linker of PBD dimer-maleimide-acetal is acid-labile.

[0225] The PBD dimer and the ADC comprising the PBD dimer can be prepared according to methods known in the art. See, for example, WO2009 / 016516, US2009 / 304710, US2010 / 047257, US2009 / 036431, US2011 / 0256157, WO2011 / 130598.

[0226] (5) Anthracycline In some embodiments, the ADC may include an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. While not intending to be bound by any particular theory, studies have shown that anthracyclines may act to kill cells by several different mechanisms, including: 1) inhibition of DNA-dependent nucleic acid synthesis by intercalation of the drug molecule into the cell's DNA; 2) production of free radicals by the drug that then react with cellular macromolecules to cause damage to the cell; and / or 3) interaction of the drug molecule with the cell membrane (see, e.g., C. Peterson et al., “Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia” in Anthracycline Antibiotics In Cancer Therapy; N.R. Bachur, “Free Radical Damage” (ibid pp. 97-102)). These potentially cytotoxic anthracyclines have been used in the treatment of a number of cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma (see, e.g., P.H-Wiernik, in Anthracycline: Current Status And New Developments, p. 11).

[0227] Non-limiting and exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and their derivatives. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (see Kratz et al., Current Med.Chem., vol.13, pp.477-523, 2006, Jeffrey et al., Bioorganic & Med.Chem.Letters, vol.16, pp.358-362, 1996, Torgov et al., Bioconj.Chem., vol.16, pp.717-721, 2005, Nagy et al., Proc.Natl.Acad.Sci.USA, vol.97, pp.829-834, 2000, Dubowchik et al., Bioorg.& Med.Chem.Letters, vol.12, pp.1529-1532, 2002, King et al., J.Med.Chem., vol.45, pp.4336-4343, 2002, EP0328147, U.S. Patent No. 6,630,579). The antibody-drug conjugate BR96-doxorubicin specifically reacts with the tumor-associated antigen Lewis-Y and has been evaluated in Phase I and Phase II trials (Saleh et al., J.Clin.Oncology, vol.18, pp.2282-2292, 2000, Ajani et al., Cancer Jour., vol.6, pp.78-81, 2000, Tolcher et al., J.Clin.Oncology, vol.17, pp.478-484, 1999).

[0228] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617, 2005). Nemorubicin is a semi-synthetic analogue of doxorubicin with a 2-methoxymorpholino group on the glycosidoamino of doxorubicin and has been clinically evaluated (Grandi et al. Cancer Treat. Rev. vol. 17, pp. 133-138, 1990, Ripamonti et al. Brit. J. Cancer, vol. 65, pp. 703-707, 1992), including phase II / III trials in hepatocellular carcinoma (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Abs1448, 2003, Quintieri, Proceedings of the American Association of Cancer Research, vol. 44: 1st Ed, Abs 4649, 2003, Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006).

[0229] Anthracyclines including PNU-159682 can be conjugated to an antibody via various linkers (US2011 / 0076287, WO2009 / 099741, US2010 / 0034837, WO2010 / 009124) containing several linking sites and the linkers described herein.

[0230] The linker of PNU-159682 maleimidacetal-Ab is acid-labile, while the linkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer(R 1 R 2 )-Ab are cleavable by proteases.

[0231] (6) Other drug moieties In addition, the drug moiety includes geldanamycin (Mandler et al., J. Nat. Cancer Inst., vol. 92, pp. 1573-1581, 2000; Mandler et al., Bioorganic & Med. Chem. Letters, vol. 10, pp. 1025-1028, 2000; Mandler et al., Bioconjugate Chem., vol. 13, pp. 786-791, 2002), as well as enzyme-active toxins and fragments thereof (including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichotheene). See, for example, WO93 / 21232.

[0232] The drug moiety also includes compounds having nuclease activity (e.g., ribonuclease or DNA endonuclease).

[0233] In certain embodiments, the immunoconjugate may contain a radioisotope. A variety of radioisotopes are available for the production of radiolabeled conjugate antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212include radioactive isotopes of Lu. In some embodiments, when an immunoconjugate is used for detection, it may include a radioactive atom for scintigraphy studies, such as Tc 99 or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be complexed to various metal chelating agents and conjugated to an antibody, for example, for PET imaging (WO2011 / 056983).

[0234] A radiolabel or other label may be incorporated into the immunoconjugate by known methods. For example, a peptide can be biosynthesized or chemically synthesized using suitable amino acid precursors that include one or more fluorine-19 atoms instead of one or more hydrogens. In some embodiments, Tc 99 I 123 Re 186 Re 188 and labels such as In 111 can be attached via cysteine residues in the antibody. In some embodiments, yttrium-90 can be attached via lysine residues in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes certain other methods.

[0235] In certain embodiments, the immunoconjugate may comprise an antibody conjugated to a prodrug activating enzyme. In some such embodiments, the prodrug activating enzyme converts a prodrug (see, e.g., peptidyl chemotherapeutic agents, WO81 / 01145) into an active drug such as an anti-cancer agent. Such immunoconjugates are useful in some embodiments in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to an antibody include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs to free drugs, arylsulfatase useful for converting sulfate-containing prodrugs to free drugs, cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil, proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsin (such as cathepsin B and L) useful for converting peptide-containing prodrugs to free drugs, D-alanyl carboxypeptidase useful for converting prodrugs containing D-amino acid substituents, carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs to free drugs, β-lactamase useful for converting drugs derivatized with β-lactam to free drugs, penicillin amidases such as penicillin V amidase and penicillin G amidase useful for converting drugs derivatized with an amine nitrogen having a phenoxyacetyl group or a phenylacetyl group to free drugs. In some embodiments, the enzyme can be covalently attached to the antibody by recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature, vol. 312, pp. 604-608, 1984.

[0236] iii) Drug loading Drug loading is represented by the average number p of drug moieties per antibody in the molecule of Formula I. The drug loading can range from 1 to 20 drug moieties (D) per antibody. The ADC of Formula I includes an aggregate of antibodies conjugated with a range of 1 to 20 drug moieties. The average number of drug moieties per antibody used in the preparation of the ADC from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC with respect to p can also be determined. In some cases, when p is a specific value different from ADCs having other drug loadings, separation, purification, and characterization of homogeneous ADCs can be achieved by means such as reverse phase HPLC or electrophoresis.

[0237] For some antibody-drug conjugates, p can be limited by the number of binding sites on the antibody. For example, as in certain of the above specific exemplary embodiments, where the binding is a cysteine thiol, the antibody may have only one or some cysteine thiol groups, or only one or some sufficiently reactive thiol groups to which a linker can be attached. In certain embodiments, at higher drug loadings, e.g., p > 5, aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates can occur. In certain embodiments, the average drug loading of the ADC ranges from 1 to about 8, about 2 to about 6, or about 3 to about 5. Indeed, for certain ADCs, it has been shown that the optimal ratio of drug moieties per antibody may be less than 8 and may be about 2 to about 5 (U.S. Patent No. 7,498,298).

[0238] In certain embodiments, during the conjugation reaction, less than the theoretical maximum of the drug moiety is conjugated to the antibody. The antibody can include, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to the drug moiety. In fact, most cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) to generate reactive cysteine thiol groups under partial or total reducing conditions. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0239] The loading (drug / antibody ratio) of the ADC can be adjusted in different ways, for example, by (i) limiting the molar excess of the drug-linker intermediate or linker reagent to the antibody, (ii) limiting the reaction time or temperature of the conjugation, and (iii) partial or limited reducing conditions for cysteine thiol modification.

[0240] It should be understood that when two or more nucleophilic groups react with the drug-linker intermediate or linker reagent, the resulting product is a mixture of ADCs having a distribution of one or more drug moieties bound to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. The individual ADCs in the mixture can be identified by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (see, for example, McDonagh et al., Prot. Engr. Design & Selection, vol. 19, pp. 299-307, 2006, Hamblett et al., Clin. Cancer Res., vol. 10, pp. 7063-7070, 2004). In certain embodiments, a homogeneous ADC having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0241] iv) A specific method for preparing an immunoconjugate The immunoconjugate, which is an ADC of formula I, can be prepared by several routes using reactions, conditions, and reagents of organic chemistry known to those skilled in the art, including: (1) reacting a nucleophilic group of the antibody with a bivalent linker reagent to form Ab-L via a covalent bond and subsequently reacting it with the drug moiety D, and (2) reacting a nucleophilic group of the drug moiety with a bivalent linker reagent to form D-L via a covalent bond and subsequently reacting it with a nucleophilic group of the antibody. An exemplary method for preparing an ADC of formula I via the latter route is described in U.S. Patent No. 7,498,298.

[0242] Nucleophilic groups on the antibody include, but are not limited to, the following: (i) the N-terminal amine group, (ii) side-chain amine groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups to which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on the linker moiety and linker reagents, including: (i) active esters such as NHS esters, HOBt esters, formates, and acid halides, (ii) alkyl and benzyl halides such as haloacetamides, and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. The antibody can be made reactive towards conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol) or TCEP (tricarbonyl ethylphosphine) so that the antibody is fully or partially reduced. Thus, each cysteine bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody, for example, through modification of lysine residues by reacting the lysine residues with 2-iminothiolane (Traut's reagent), and the amine can be converted to a thiol. Also, reactive thiol groups can be introduced into the antibody by introducing one, two, three, four, or more cysteine residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine amino acid residues).

[0243] The antibody-drug conjugates of the present invention can also be generated by the reaction between an electrophilic group on an antibody or antibody fragment, such as an aldehyde group or a ketone carbonyl group, and a nucleophilic group on a linker reagent or a drug. Useful nucleophilic groups on the linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on the linker reagent or the drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidation reagent to form an aldehyde or ketone group that can react with an amine group of the linker reagent or drug moiety. The resulting imine Schiff base can form a stable bond or can be reduced, for example, with a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate portion of the glycosylated antibody with either galactose oxidase or sodium metaperiodate can result in a carbonyl (aldehyde and ketone) group in the antibody that can react with a suitable group on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can react with sodium metaperiodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, Bioconjugate Chem., vol. 3, pp. 138-146, 1992, U.S. Patent No. 5,362,852). Such aldehydes can react with the drug moiety or linker nucleophile.

[0244] Exemplary nucleophilic groups on the drug moiety include, but are not limited to, amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazide groups that can react to form a covalent bond with an electrophilic group on the linker moiety, as well as the following linker reagents: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl halides and benzyl halides such as haloacetamides, (iii) aldehydes, ketones, carboxyls, and maleimide groups.

[0245] Non-limiting and exemplary crosslinking reagent that can be used to prepare ADCs are described in the section entitled "Exemplary Linkers" herein. Methods of using such crosslinking reagents to link two moieties, including a proteinaceous moiety and a chemical moiety, are known in the art. In some embodiments, a fusion protein comprising an antibody and a cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule may comprise a region encoding an antibody and a region encoding the cytotoxic portion of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0246] In yet another embodiment, an antibody or antibody fragment can be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting of tumors, and the antibody / antibody fragment-receptor conjugate is administered to a patient, followed by using a clearing agent to remove unbound conjugate from the circulation, and then administering a "ligand" (such as avidin) that is conjugated to a cytotoxic agent (such as a drug or a radioactive nucleotide).

[0247] C. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-CTLA4 antibodies or antibody fragments provided herein can be used to detect the presence of CTLA4 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues such as cells or tissues of the breast, pancreas, esophagus, lung and / or brain.

[0248] A further aspect of the invention relates to the anti-CTLA4 antibodies or antibody fragments of the invention for diagnosing and / or monitoring cancer or another disease in which the CTLA4 expression level is increased or decreased from the normal physiological level at at least one location within the body.

[0249] In preferred embodiments, the antibodies or antibody fragments of the invention can be labeled with a detectable molecule or substance such as a fluorescent molecule, a radioactive molecule, or any other label known in the art as described above. For example, the antibodies or antibody fragments of the invention can be labeled with a radioactive molecule. For example, suitable radioactive molecules include, but are not limited to, 123 I, 124 I, 111 In, 186 Re, and 188 radioactive atoms used in scintigraphy studies such as Re. Also, the antibodies or antibody fragments of the invention can be labeled with spin labels for nuclear magnetic resonance (NMR) imaging such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound imaging.

[0250] The antibody or antibody fragment of the present invention may be useful for the diagnosis and staging of cancers and diseases associated with overexpression of CTLA4. Cancers associated with overexpression of CTLA4 include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioma tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, sarcoma, blood cancer (leukemia), astrocytoma, as well as various types of head and neck cancers, or other hyperproliferative diseases that express or overexpress CTLA4.

[0251] The antibody or antibody fragment of the present invention may be useful for the diagnosis of diseases other than cancers in which CTLA4 expression is increased or decreased. For such diagnosis, both soluble and cellular CTLA4 forms can be used. Typically, such diagnostic methods involve the use of a biological sample obtained from a patient. A biological sample encompasses various sample types obtained from a subject that can be used in a diagnostic or monitoring assay. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. For example, a biological sample includes cells obtained from a tissue sample collected from an individual suspected of having a cancer associated with CTLA4 overexpression, and in a preferred embodiment, cells obtained from glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer. Biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0252] In certain embodiments, the present invention is a method for diagnosing a cancer associated with CTLA4 overexpression in a subject by detecting CTLA4 on cells from the subject using an antibody of the present invention. In particular, the method comprises the following steps: (a) contacting a biological sample of the subject with an antibody or antibody fragment according to the present invention under conditions suitable for the antibody or antibody fragment to form a complex with cells in the biological sample expressing CTLA4; (b) detecting and / or quantifying the complex, wherein detecting the complex indicates cancer associated with overexpression of CTLA4, and detecting and / or quantifying.

[0253] To monitor cancer progression, the method according to the invention may be repeated at different times to determine whether antibody binding to the sample increases or decreases, from which it is possible to determine whether the cancer is progressing, regressing or stabilizing.

[0254] In certain embodiments, the invention is a method of diagnosing a disease associated with expression or overexpression of CTLA4, or a decrease or increase in a soluble form of CTLA4. Examples of such diseases can include human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases.

[0255] In one embodiment, an anti-CTLA4 antibody or antibody fragment for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of CTLA4 in a biological sample is provided. In a further aspect, a method of quantifying the amount of CTLA4 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-CTLA4 antibody or antibody fragment as described herein under conditions that permit binding of the anti-CTLA4 antibody or antibody fragment to CTLA4, and detecting whether a complex is formed between the anti-CTLA4 antibody or antibody fragment and CTLA4. Such methods may be performed in vitro or in vivo. In one embodiment, the anti-CTLA4 antibody or antibody fragment is used to select a subject eligible for treatment. In some embodiments, treatment comprises administering an anti-CTLA4 antibody or antibody fragment to the subject.

[0256] In certain embodiments, a labeled anti-CTLA4 antibody or antibody fragment is provided. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radiolabels), as well as moieties such as enzymes or ligands that are indirectly detectable (e.g., by an enzymatic reaction or molecular interaction). Exemplary labels include, but are not limited to, radioisotopes ( 32 P, 14 C, 125 I, 3 H, and 131 I), or fluorophores (rare earth chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.), luciferase (e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (uricase, xanthine oxidase, etc.), coupled enzymes that oxidize a dye precursor using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, bacteriophage labels, stable free radicals, and the like.

[0257] D. Pharmaceutical Formulations The anti-CTLA4 antibody or antibody fragment has cell killing activity. This cell killing activity extends to multiple different types of cell lines. Further, once these antibodies or antibody fragments are conjugated to a cytotoxic agent, they can reduce tumor size and may exhibit reduced toxicity. Thus, anti-CTLA4 antibodies, fragments thereof, or immunoconjugates may be useful in the treatment of proliferative diseases associated with CTLA4 expression. The antibodies, fragments, or immunoconjugates can be used alone or in combination with any suitable agent or other conventional therapy.

[0258] Diseases associated with CTLA4 expression, overexpression or activation can be treated using an anti-CTLA4 antibody or antibody fragment. There is no particular limitation on the type of cancer or tissue that can be treated other than the requirement for CTLA4 expression. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioma tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer. More preferred cancers are glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer.

[0259] Anti-CTLA4 antibodies or antibody fragments are potential activators of the innate immune response and can thus be used in the treatment of human immune diseases such as sepsis. Also, the anti-CTLA4 antibodies or antibody fragments of the present invention can be used as adjuvants for immunization such as vaccines, and can also be used, for example, as anti-infective agents against bacteria, viruses and parasites.

[0260] An anti-CTLA4 antibody or antibody fragment can be used to protect against, prevent or treat thrombotic diseases such as venous thrombosis, arterial thrombosis and atherosclerotic thrombosis. An anti-CTLA4 antibody or antibody fragment can be used to protect against, prevent or treat cardiovascular diseases, and can also be used to prevent or suppress the entry of viruses such as Lassa virus and Ebola virus and to treat viral infections.

[0261] In each of the embodiments of the treatment methods described herein, the anti-CTLA4 antibody, antibody fragment, or immunoconjugate of an anti-CTLA4 antibody or antibody fragment can be delivered in a manner consistent with conventional methods relevant to the management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody, antibody fragment or immunoconjugate is administered to a subject in need thereof under conditions and for a time sufficient to prevent or treat the disease or disorder. Accordingly, one aspect of the invention relates to a method for treating a disease associated with the expression of CTLA4, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, antibody fragment, or immunoconjugate of the invention.

[0262] In the case of administration, the anti-CTLA4 antibody, antibody fragment, or immunoconjugate can be formulated as a pharmaceutical composition. A pharmaceutical composition comprising an anti-CTLA4 antibody, antibody fragment, or immunoconjugate can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution, or composition comprising a pharmaceutically acceptable carrier.

[0263] A pharmaceutically acceptable carrier is a substance that can be tolerated by the recipient patient. Sterile phosphate buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington’s Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further comprise one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

[0264] The form, route of administration, dosage, and regimen of the pharmaceutical composition will of course depend on the condition being treated, the severity of the disease, the age, weight, and sex of the patient, etc. These considerations can be taken into account by those skilled in the art in formulating a suitable pharmaceutical composition. The pharmaceutical composition of the present invention can be formulated for topical administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration, etc.

[0265] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may in particular be isotonic sterile physiological saline (such as monosodium phosphate or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or a mixture of such salts), or may be a dry (in particular, lyophilized) composition, for example enabling the constitution of an injectable solution upon addition of sterile water or physiological saline.

[0266] In some embodiments, tonicity agents, sometimes also known as "stabilizers", are present to adjust or maintain the tonicity of the liquid in the composition. When used with highly charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can reduce the potential for intermolecular and intramolecular interactions by interacting with the charged groups of the amino acid side chains. Tonicity agents can be present in any amount from 0.1 to 25% by weight, preferably 1 to 5% by weight, of the pharmaceutical composition. Preferred tonicity agents include polyhydric sugar alcohols, preferably sugar alcohols with three or more hydroxyl groups such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0267] Examples of additional excipients include agents that can function as one or more of the following: (1) bulking agents, (2) solubilizing agents, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above), amino acids (e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine), organic sugars or sugar alcohols (e.g., sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inositol, myo-inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol), sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-thioglycerol and sodium thiosulfate), low molecular weight proteins (e.g., human serum albumin, bovine serum albumin, gelatin or other immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), monosaccharides (e.g., xylose, mannose, fructose, glucose), disaccharides (e.g., lactose, maltose, sucrose), trisaccharides (e.g., raffinose) and polysaccharides (e.g., dextrin or dextran) may be mentioned.

[0268] Nonionic surfactants or detergents (also known as "wetting agents") can be used to assist in solubilizing the therapeutic agent and to protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface loading without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0269] Suitable nonionic surfactants include polysorbates (such as 20, 40, 60, 65, 80), polyoxamers (such as 184, 188), PLURONIC® polyols, TRITON® , polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Usable anionic detergents include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0270] The dosage used for administration is adapted as a function of various parameters, in particular as a function of the mode of administration used, as a function of the associated pathology, or alternatively as a function of the desired treatment period. To prepare the pharmaceutical composition, an effective amount of the antibody or antibody fragment can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0271] Dosage forms suitable for injection include sterile aqueous solutions or dispersions, preparations containing sesame oil, peanut oil or aqueous propylene glycol solutions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injectable. It must be stable under the conditions of manufacture and storage, and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0272] Solutions of the active compounds as free bases or as pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant. The dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0273] The anti-CTLA4 antibody or antibody fragment can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid), or such organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid). Also, salts formed with free carboxyl groups can be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide), and such organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine).

[0274] The carrier can also be a solvent or dispersion medium including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to contain isotonic agents, e.g., sugars or sodium chloride. Sustained absorption of injectable compositions can be brought about by the use in the composition of agents that delay absorption, e.g., aluminum monostearate and gelatin.

[0275] Sterile injectable solutions are prepared by incorporating the required amount of the active compound into a suitable solvent containing one or more additional ingredients other than those enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle, containing a basic dispersion medium and other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously filter-sterilized solution thereof.

[0276] Also, for direct injection, the preparation of more solution, or a higher concentration solution, is contemplated, and the use of dimethyl sulfoxide (DMSO) as a solvent is expected to result in very rapid penetration and the delivery of a high concentration of the active agent to small tumor areas.

[0277] Upon formulation, the solutions are administered in a manner compatible with the dosage formulation and in therapeutically effective amounts. The formulations are readily administered in a variety of dosage forms such as those of the injectable solutions described above, although drug release capsules and the like can also be used.

[0278] In the case of parenteral administration in an aqueous solution, the solution is preferably buffered as required and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this context, sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure. For example, a single dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous drip solution or injected into the proposed injection site (see, e.g., “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variations in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0279] An antibody or antibody fragment may be formulated in a therapeutic mixture to deliver from about 0.0001 to 10.0 mg, or about 0.001 to 5 mg, or about 0.001 to 1 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0, or even about 10 mg per single dose. Multiple administrations can also be carried out at selected time intervals.

[0280] In addition to compounds formulated for parenteral administration such as intravenous or intramuscular injection, other pharmaceutically acceptable dosage forms include, for example, tablets or other solids for oral administration, sustained release capsules, and any other dosage forms currently in use.

[0281] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for introducing an antibody or antibody fragment into a host cell. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0282] Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (sizes around 0.1 μm) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be readily prepared.

[0283] Liposomes are dispersed in an aqueous medium and are formed from phospholipids that spontaneously form multilamellar concentric bilayer vesicles (also referred to as multilamellar vesicles (MLV)). MLV generally have a diameter of 25 nm to 4 μm. Sonication of MLV forms small unilamellar vesicles (SUV) in the range of 200 to 500 Å in diameter that contain an aqueous solution within the core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0284] The pharmaceutical preparation containing the anti-CTLA4 antibody or antibody fragment described in this specification is in the form of a lyophilized preparation or an aqueous solution, and such an antibody or antibody fragment having the desired purity is prepared by mixing it with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include, but are not limited to, buffers (such as phosphoric acid, citric acid, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzylammonium chloride), hexamethonium chloride, benzalkonium chloride, benzetonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides (less than about 10 residues), proteins (such as serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin), chelating agents (such as EDTA), saccharides (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn protein complexes), and / or nonionic surfactants (such as polyethylene glycol (PEG)).

[0285] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGP and methods of use including rHuPH20 are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0286] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation containing a histidine - acetate buffer.

[0287] The formulations herein may also, if desired, contain two or more active ingredients for a particular indication being treated. Preferably, components having complementary activities that do not adversely affect each other may be combined in a single formulation. For example, in addition to the anti - CTLA4 antibodies, antibody fragments or immunoconjugates of the present invention, it may be desirable to provide an EGFR antagonist (such as erlotinib), an anti - angiogenic agent (such as a VEGF antagonist, which may be an anti - VEGF antibody), or a chemotherapeutic agent (such as a taxoid or a platinum agent). Such active ingredients are preferably present in combination in an amount effective for the intended purpose.

[0288] In one embodiment, the anti-CTLA4 antibody, antibody fragment or immunoconjugate of the present invention is combined in a formulation with another antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins (including WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16). The combination may be in the form of two separate molecules, i.e., the anti-CTLA4 antibody, antibody fragment or immunoconjugate of the present invention and another antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule, having binding affinity for both CTLA4 and other antigens, and thus forming a multispecific (e.g., bispecific) antibody.

[0289] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization. For example, hydroxyethylmethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules may be used in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0290] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include a semipermeable matrix of a solid hydrophobic polymer containing the antibody or antibody fragment, and the matrix can be in the form of a shaped article, such as a film or a microcapsule.

[0291] Formulations used for in vivo administration are generally sterile. Sterilization can be readily achieved, for example, by filtration through a sterilizing filter membrane.

[0292] E. Treatment Methods and Compositions Any of the anti-CTLA4 antibodies or antibody fragments provided herein can be used in a treatment method. In one aspect, an anti-CTLA4 antibody or antibody fragment for use as a medicament is provided. In a further aspect, an anti-CTLA4 antibody or antibody fragment for use in the treatment of cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, thyroid cancer, liver cancer, osteosarcoma, and / or melanoma) is provided. In certain embodiments, an anti-CTLA4 antibody or antibody fragment for use in a treatment method is provided. In certain embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having cancer, comprising administering to the individual an effective amount of the anti-CTLA4 antibody or antibody fragment. In certain embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having an immune disorder (e.g., autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) or diabetes, comprising administering to the individual an effective amount of the anti-CTLA4 antibody or antibody fragment. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., those described below). In further embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function.

[0293] In certain embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function in an individual, and administering to the individual an effective amount of the anti-CTLA4 antibody or antibody fragment to inhibit angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), development of tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function. An "individual" according to any of the above embodiments is preferably a human.

[0294] In a further aspect, the present invention provides the use of an anti-CTLA4 antibody or antibody fragment in the manufacture or preparation of a medicament. In one embodiment, the medicament is a medicament for the treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, thyroid cancer, liver cancer, osteosarcoma, and / or melanoma). In a further embodiment, the medicament is a medicament for use in a method of treating cancer, which comprises administering to an individual having cancer an effective amount of the medicament. In a further embodiment, the medicament is a medicament for use in a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus) or diabetes, which comprises administering to an individual an effective amount of an anti-CTLA4 antibody or antibody fragment. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., those described below). In a further embodiment, the medicament is a medicament for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function. In a further embodiment, the medicament is a medicament for use in a method of inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function in an individual, and administering to the individual an effective amount of the medicament to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce a section of inflammatory cytokines (e.g., derived from tumor-associated macrophages), inhibit the development of tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or inhibit tumor stromal function. The "individual" according to any of the above embodiments may be a human.

[0295] In a further aspect, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an anti-CTLA4 antibody or antibody fragment. In such one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., those described below). The "individual" according to any of the above embodiments may be a human.

[0296] In a further aspect, the present invention provides a method for treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes. In such one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., those described below). The "individual" according to any of the above embodiments may be a human.

[0297] In a further aspect, the present invention provides a method for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., derived from tumor-associated macrophages), tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or tumor stromal function in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA4 antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce the section of inflammatory cytokines (e.g., derived from tumor-associated macrophages), inhibit the development of tumor vascular structure (e.g., intratumoral vascular structure or tumor-associated vascular structure), and / or inhibit tumor stromal function. In one embodiment, the "individual" is a human.

[0298] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the anti-CTLA4 antibodies or antibody fragments provided herein for use, for example, in any of the treatment methods described above. In one embodiment, the pharmaceutical formulation comprises any of the anti-CTLA4 antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-CTLA4 antibodies or antibody fragments provided herein and at least one additional therapeutic agent, for example, as described below.

[0299] In each of the treatments described above and in each treatment, the antibody or antibody fragment of the present invention can be used alone, as an immunoconjugate, or in combination with other agents during treatment. For example, the antibody of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, such as bevacizumab). In certain embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cell division inhibitor. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatin). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0300] Such combination therapies described above include co-administration (where two or more therapeutic agents are included in the same or separate formulations), as well as separate administration, in which case administration of the antibody or antibody fragment can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment can also be used in combination with radiation therapy.

[0301] An anti-CTLA4 antibody or antibody fragment can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to the physician. The antibody or antibody fragment, although not necessarily required, is optionally formulated with one or more drugs currently being used to prevent or treat the disorder in question. The effective amount of such other drug depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dosage and by the same route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and by any route determined to be empirically / clinically appropriate.

[0302] For the prevention or treatment of a disease, the appropriate dosage of an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody or antibody fragment, and the discretion of the attending physician. The antibody or antibody fragment is preferably administered to the patient once or over a series of treatments. Depending on the type and severity of the disease, about 1 μg of antibody or antibody fragment / kg of patient body weight to 40 mg of antibody or antibody fragment / kg of patient body weight can be a first candidate dosage for administration to the patient, whether by, for example, one or more separate administrations or by continuous infusion. One typical daily dosage can range from about 1 μg of antibody or antibody fragment / kg of patient body weight to over 100 mg of antibody or antibody fragment / kg of patient body weight, depending on the factors described above. In the case of repeated administration over several days, depending on the condition, the treatment will generally continue until the desired suppression of the disease symptoms occurs. Such dosages can be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about 2 to about 20 doses, or for example, about 6 doses of the antibody or antibody fragment). Higher loading doses than the first dose, followed by one or more lower doses, can be administered. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0303] The specific dosage of the anti-CTLA4 antibody or antibody fragment of the present invention that can be administered for the prevention or treatment of a target disease can be about 0.3, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, 9.0, 9.6, or 10.2 mg of antibody or antibody fragment / kg of patient body weight. In certain embodiments, the dosage can range from 0.3 to 2.4, 2.4 to 4.2, 4.2 to 6.0, 6.0 to 7.8, 7.8 to 10.2, 10.2 to 12, 12 to 14, 14 to 16, 16 to 18, or 18 to 20 mg of antibody or antibody fragment / kg of patient body weight. When administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment, or as an immunoconjugate, the dosage of the antibody or antibody fragment remains the same. Further, a polypeptide having anti-CTLA4 activity is administered in the same amount as the antibody or antibody fragment.

[0304] The single dose of the pharmaceutical preparation of the present invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the present invention from about 45 μg of antibody or antibody fragment, from about 45 μg of antibody or antibody fragment from about 13,600 mg, or from about 45 μg of antibody or antibody fragment from about 5440 mg. In some embodiments, the single dose of the pharmaceutical preparation of the present invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the present invention in an amount of 135 mg to 1,387 mg, or amounts such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg. In certain embodiments, the amount of the anti-CTLA4 antibody or antibody fragment of the present invention in a single dose of the pharmaceutical preparation ranges from 135 to 235, 235 to 335, 335 to 435, 435 to 535, 535 to 635, 635 to 735, 735 to 835, 835 to 935, 935 to 1035, 1035 to 1135, 1135 to 1235, 1235 to 1387 mg. The amount of antibody or antibody fragment in a single dose of the pharmaceutical preparation remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor, or as an immunoconjugate, or in combination with another antibody or antibody fragment against another antigen disclosed herein. Further, a polypeptide having anti-CTLA4 activity will be included in a single dose of the pharmaceutical preparation in the same amount as the antibody or antibody fragment.

[0305] In one example, the anti-CTLA4 antibody or antibody fragment can be conjugated to another immune checkpoint inhibitor molecule or can form part of a bispecific antibody with another immune checkpoint inhibitor.

[0306] The other immune checkpoint inhibitor molecule can be an antibody or antibody fragment against another immune checkpoint other than CTLA4. The combination can be the anti-CTLA4 antibody or antibody fragment disclosed in the present application and another immune checkpoint inhibitor molecule administered as a separate molecule or as a bispecific antibody. Such a bispecific antibody has binding activity to CTLA4 and a second binding activity to another immune checkpoint.

[0307] Immune checkpoints can be selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences, vol. 20, 158, 2019). Further immune checkpoints include B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS (Manni et al., Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment, Bbacan, https: / / doi.org / 10.1016 / j.bbcan.2018.12.002, 2018).

[0308] The immune checkpoint is preferably PD-1 or PD-L1.

[0309] It should be understood that any of the above formulations or treatment methods can be carried out using the antibody fragment or immunoconjugate of the present invention instead of, or in addition to, an anti-CTLA4 antibody.

[0310] Enhancing the host immune function to fight tumors is an area of increasing interest. Conventional methods include (i) enhancing APCs, for example, (a) injecting DNA encoding allogeneic MHC antigens into the tumor, or (b) transfecting biopsy tumor cells with genes that increase the probability of immune antigen recognition in the tumor (e.g., immune-stimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), (iii) adoptive cell immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cell immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, for example, through stimulation with IL-2 or the tumor or both. Additionally, isolated T cells that are dysfunctional can also be activated by in vitro application of the anti-PD-L1 antibody of the present invention. The thus-activated T cells may then be readministered to the host. One or more of these methods can be used in combination with the administration of the antibodies, antibody fragments, or immunoconjugates of the present invention.

[0311] Traditional therapies for cancer include the following: (i) radiotherapy (e.g., radiation therapy, X-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiotherapy can be administered via external beam radiotherapy (EBRT) or internally via brachytherapy, (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells, (iii) targeted therapy, or drugs that specifically affect deregulated proteins in cancer cells (e.g., imatinib, gefitinib, tyrosine kinase inhibitors; monoclonal antibodies, photodynamic therapy), (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines), (v) hormone therapy, or blocking hormones (e.g., if the tumor is hormone-sensitive), (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth, and (vii) palliative care, or treatment aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant can enable a more aggressive treatment regimen.

[0312] In the treatment of cancer, any of the aforementioned conventional treatments for cancer immunotherapy can be performed before, after, or simultaneously with the administration of an anti-CTLA4 antibody or antibody fragment. In addition, the anti-CTLA4 antibody or antibody fragment can be administered before, after, or simultaneously with conventional cancer treatments such as the administration of a tumor-binding antibody (e.g., a monoclonal antibody, a toxin-conjugated monoclonal antibody) and / or the administration of a chemotherapeutic agent.

[0313] F. Products and Kits In another aspect of the invention, there is provided a product comprising an anti-CTLA4 antibody or antibody fragment and other materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The product includes a container and a label or package insert provided in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, and the like. The container can be formed from various materials such as glass or plastic. The container can hold the composition by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous infusion bag or a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody or antibody fragment of the invention. The label or package insert indicates that the composition is used for treating a selected condition. Further, the product may include (a) a first container in which the composition is contained and the composition contains an antibody or antibody fragment, and (b) a second container in which the composition is contained and the composition contains a further cytotoxic agent or other therapeutic agent. The product in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0314] It should be understood that any of the above products can include the immunoconjugate of the invention instead of or in addition to the anti-CTLA4 antibody or antibody fragment.

[0315] Finally, the present invention also provides a kit comprising at least one antibody or antibody fragment of the present invention. Kits comprising the polypeptides, antibodies or antibody fragments, or antibody-drug conjugates of the present invention are useful for the detection of CTLA4 expression (increase or decrease), or for therapeutic or diagnostic assays. Kits of the present invention may comprise an antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided that contain antibodies for detecting and quantifying CTLA4 in vitro (e.g., ELISA or Western blot). Such antibodies useful for detection can be provided with a label, such as a fluorescent or radiolabel.

[0316] The kit further comprises instructions regarding their use. In some embodiments, the instructions include the instructions required by the US Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kit further comprises instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of CTLA4 in the sample. In some embodiments, the kit comprises one or more antibodies or antibody fragments. In other embodiments, the kit further comprises one or more enzymes, enzyme inhibitors or enzyme activators. In still other embodiments, the kit further comprises one or more chromatography compounds. In still other embodiments, the kit further comprises one or more compounds used to prepare a sample for a spectroscopic assay. In further embodiments, the kit further comprises a comparative reference material for interpreting the presence or absence of CTLA4 according to the intensity of an indicator, color spectrum, or other physical attribute.

[0317] The following examples are illustrative of the soft gelatin capsules of the present disclosure, but are not limiting. Other suitable modifications and adaptations of the various conditions and parameters commonly encountered in the art and obvious to those of ordinary skill in the art are within the scope of the present disclosure.

Example

[0318] Example 1: Conditionally Active Biological (CAB) Antibody Against CTLA4 In this example, antibodies against CTLA4 were produced (Table 2).

Table 2

[0319] These anti-CTLA4 antibodies were further characterized. Data for antibodies BA-087-05-19 and BA-087-08-32 are presented in this application.

[0320] Example 2: ELISA assay for the binding activity of anti-CTLA4 antibodies The binding activities of BA-087-05-19 and BA-087-08-32 to immobilized recombinant human CTLA4 were determined using an enzyme-linked immunosorbent assay (ELISA) in a buffer at pH 6.0 (the pH of the tumor microenvironment) or a buffer at pH 7.4 (normal physiological pH). Serial dilutions of BA-087-05-19 and BA-087-08-32 were bound to the extracellular domain of recombinant human CTLA4 cells immobilized in wells. The amounts of bound BA-087-05-19 and BA-087-08-32 were quantified using an anti-human IgG antibody conjugated to horseradish peroxidase (HRP), and then reacted with a 3,3’,5,5’-tetramethylbenzidine (TMB) colorimetric substrate to produce a colored product. The OD absorbance in each well was proportional to the amount of bound BA-087-05-19 and BA-087-08-32. The EC 50 values for binding to human CTLA4 at pH 6.0 were calculated using a non-linear fitting model (variable slope, 4 parameters) of GraphPad Prism version 7.03.

[0321] Table 3-4 shows the EC 50Values are shown, and the binding curves of representative experiments are shown in FIGS. 3A - 3B. Both BA - 087 - 05 - 19 and BA - 087 - 08 - 32 showed similar binding activity to human CTLA4 at pH 6.0 and significantly decreased binding activity at pH 7.4 compared to ipilimumab and ipilimumab analogs. [Table 3] [Table 4]

[0322] In addition, the binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 to immobilized recombinant cynomolgus CTLA4 extracellular domain were also determined by ELISA. Table 5 shows the EC 50 of the binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 to cynomolgus CTLA4 at pH 6.0. FIGS. 4A - 4B show the binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 to cynomolgus CTLA4 at pH 6.0 and pH 7.4. [Table 5]

[0323] The EC 50 of the binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 at pH 6.0 of the tumor microenvironment measured by ELISA was found to be 8.18 ng / mL and 9.78 ng / mL, respectively, for human CTLA4, and the EC 50It was the same. BA-087-05-19 has similar binding activities to both human CTLA4 and cynomolgus monkey CTLA4 at pH 6.0, while BA-087-08-32 has a decreased binding activity to cynomolgus monkey CTLA4 compared to its binding activity to human CTLA4 at pH 6.0. The decrease in the binding activity of BA-087-08-32 to cynomolgus monkey CTLA4 at pH 6.0 as seen in ELISA seems to be specific to the ELISA assay since the same decrease was not observed using either SPR or FACS. The binding activities of BA-087-05-19 and BA-087-08-32 to human CTLA4 or cynomolgus monkey CTLA4 at normal physiological pH 7.4 measured by ELISA were significantly lower than the binding activities at pH 6.0.

[0324] Example 3: pH-Dependent Binding Activity of Anti-CTLA4 Antibodies The binding activities of the antibodies to CTLA4 were tested using an ELISA assay in the pH range of 5.0 to 7.4. Recombinant human CTLA4 extracellular domain was immobilized in wells in the range of pH buffer (pH 5.0 to pH 7.4) to mimic the pH of the tumor microenvironment (pH 5.5 to pH 6.7) and normal physiological pH (pH 7.4), and the binding activities were measured using ELISA. Antibodies BA-087-05-19 and BA-087-08-32 were serially diluted and their binding activities to the recombinant human CTLA4 extracellular domain were measured. The amounts of bound antibodies BA-087-05-19 and BA-087-08-32 were quantified using an anti-human IgG antibody conjugated to horseradish peroxidase (HRP), and then reacted with a 3,3’,5,5’-tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The OD absorbance in each well was proportional to the amounts of bound BA-087-05-19 and BA-087-08-32.

[0325] The pH inflection point of BA-087-05-19 (= 50% of the binding activity at pH 6.0) was calculated to be pH 6.97, and at pH 6.66, 90% of the binding activity was present. The pH inflection point of BA-087-08-32 (= 50% of the binding activity at pH 6.0) was calculated to be pH 6.43, and at pH 6.2, 90% of the binding activity was present.

[0326] The average OD values (from two replicates) at different pHs were plotted against the pH of the buffer using Softmax Pro software (Molecular Devices). Curve fitting was performed using a four-parameter model incorporated into the software. The inflection point of the pH curve (= 50% of the binding activity at pH 6.0) is equal to parameter C of the fitting equation. The binding activity at pH 6.0 was set to 100%. The pH for 90% binding activity was interpolated from the fitted curve using the "InterpX" function of Softmax Pro software.

[0327] The average pHs for 50% and 90% activities of BA-087-05-19 and BA-087-08-32 were calculated using the pH values obtained in Experiments 1 - 4. In Experiments 1 - 4, BA-087-05-19 (lot number #6972) and BA-087-08-32 (lot number #6978) were used. In Experiment 5, other lots of BA-087-05-19 (lot number #6901) and BA-087-08-32 (lot number #6902) were used. The pHs for 50% and 90% activities of BA-087-05-19 and BA-087-08-32 determined from the data of Experiment 5 were similar to the average pH values calculated using the pH values of Experiments 1 - 4. See Table 6.

Table 6

[0328] Figure 5 shows the binding activities of BA-087-05-019 and BA-087-08-32 to recombinant human CTLA4 in various pH buffers, as well as the positive control ipilimumab and ipilimumab analogs. The inflection points of pH-dependent binding for BA-087-05-19 and BA-087-08-32 were calculated to be pH 6.97 and pH 6.43, respectively. The 90% binding activities for BA-087-05-19 and BA-087-08-32 were present at pH 6.34 and pH 6.2, respectively. In addition, weaker binding activities were detected for both BA-087-05-19 and BA-087-08-32 at normal physiological pH of 7.4 (Figure 5).

[0329] Example 4: Binding Kinetics of Anti-CTLA4 Antibodies The binding kinetics of antibodies BA-087-05-19 and BA-087-08-32 were measured using surface plasmon resonance (SPR) on immobilized recombinant human CTLA4 or cynomolgus monkey CTLA4 at pH 6.0 and pH 7.4. The CTLA4 extracellular domain (human or cynomolgus monkey) was immobilized on the surface of the sensor chip. Different concentrations of BA-087-05-19 and BA-087-08-32 were injected, and the binding interactions with the immobilized CTLA4 and control surfaces were monitored in real time. The binding kinetics were calculated using a 1:1 Langmuir model incorporated into the analysis software.

[0330] Antibody BA-087-05-19 showed binding activity at sub-nanomolar concentrations at pH 6.0. The binding activity decreased by approximately 2-fold from pH 6.0 to pH 7.4 (K D [pH6.0]=0.5nM, K D [pH7.4]=1.1nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 reached only approximately 20% of the signal level detected at pH 6.0, indicating that only a small portion of the BA-087-05-19 present was able to bind to human CTLA4 at pH 7.4. See Table 7.

Table 7

[0331] Moreover, BA-087-08-32 exhibited binding activity at sub-nanomolar concentrations at pH 6.0. The binding activity decreased by approximately 100-fold from pH 6.0 to pH 7.4 (K D [pH6.0] = 0.45 nM, K D [pH7.4] = 45 nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 reaches only about 10% of the signal level at pH 6.0, indicating that only a very small fraction of the BA-087-08-32 present can bind to human CTLA4 at pH 7.4. See Table 8.

Table 8

[0332] The commercially available anti-CTLA4 antibody ipilimumab (Yervoy (trademark)) was used as a control under the same conditions, and the binding activities were found to be very similar at pH 6.0 and pH 7.4 (K D [pH6.0] = 1.39 nM, K D [pH7.4] = 1.37 nM). Therefore, the binding activity of ipilimumab was not pH-dependent. See Table 9. Also, the SPR signals obtained were also very similar at both pH 6.0 and pH 7.4.

Table 9

[0333] The binding activities of antibodies BA-087-05-19, BA-087-08-32, and ipilimumab to cynomolgus CTLA4 were tested for human CTLA4 using the same conditions as described above. All three antibodies had a fast off-rate, and the SPR signal reached equilibrium at all antibody concentrations tested. K D was calculated by plotting the maximum SPR signal at each concentration against the antibody concentration. The experiment was performed three times at each pH.

[0334] Antibody BA-087-05-19 binds to cynomolgus monkey CTLA4 with a K of 1.96 nM at pH 6.0. At pH 7.4, the calculated value shows a K D >100 nM. Antibody BA-087-08-32 binds to cynomolgus monkey CTLA4 with a K of 5.95 nM at pH 6.0. At pH 7.4, the obtained SPR signal is too low to calculate a K D . Antibody ipilimumab binds to cynomolgus monkey CTLA4 with a K of 6.58 nM at pH 6.0 and a K of 6.80 nM at pH 7.4. D D D、 D

[0335] Example 5: FACS Analysis of Anti-CTLA4 Antibodies The binding activities of antibodies BA-087-05-19 and BA-087-08-32 to human CTLA4 and cynomolgus monkey CTLA4 expressed on the cell surface of CHO cells in a buffer at pH 6.0 or pH 7.4 were measured by FACS. Serial dilutions of BA-087-05-19, BA-087-08-32, ipilimumab, and ipilimumab analogs were added to CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4. The amount of antibody bound to the cells was quantified using an anti-human IgG antibody conjugated to a fluorophore. The EC 50 values for binding to the cells at pH 6.0 and 7.4 were calculated using a non-linear fitting (variable slope, 4-parameter) model incorporated in GraphPad Prism software (version 7.03). The expression levels of human CTLA4 or cynomolgus monkey CTLA4 on the surface of CHO cells were determined using the BD QuantiBRITE™ PE kit.

[0336] ​​​​Using each of the cell lines, at least two independent duplicate FACS experiments were performed for each antibody. Figures 6A - 6B show the binding activity of the antibodies to human CTLA4 on CHO cells (CHO - huCTLA4) at pH 6.0 and 7.4. Figures 7A - 7B show the binding activity of the antibodies to cynomolgus CTLA4 on CHO cells (CHO - cynoCTLA4) at pH 6.0 and 7.4. The binding activities at different antibody concentrations are plotted in these figures.

[0337] The binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 at pH 6.0 measured by FAC were found to have mean EC of 350.1 and 243.4 ng / mL for human CTLA4, and 316.2 and 402.6 ng / mL for cynomolgus CTLA4, respectively. 50 The binding activities of ipilimumab and ipilimumab analogs at pH 6.0 measured by FAC were found to have mean EC of 341.1 ng / mL and 325.4 ng / mL for human CTLA4, and 337.5 ng / mL and 319.6 ng / mL for cynomolgus CTLA4, respectively. 50 Even at the highest concentration tested, the binding activities of BA - 087 - 05 - 19 and BA - 087 - 08 - 32 at pH 7.4 were weaker than those at pH 6.0.

[0338] Both BA - 087 - 05 - 19 and BA - 087 - 08 - 32 bind to human CTLA4 and cynomolgus CTLA4 with similar affinities as ipilimumab and ipilimumab analogs at pH 6.0. However, BA - 087 - 05 - 19 and BA - 087 - 08 - 32 have much weaker binding activities to human and cynomolgus CTLA4 at pH 7.4 compared to the binding activities of ipilimumab and ipilimumab analogs at pH 7.4. No binding activity was detected for CHO cells that did not express CTLA4.

[0339] Finally, the saturation of antibodies on CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4 at pH 7.4 was also measured by FACS (Figures 8A - 8B). At pH 7.4, antibodies BA - 087 - 05 - 19 and BA - 087 - 08 - 32 showed less binding to CHO cells compared to the control ipilimumab analog.

[0340] Example 6: ELISA and FACS Analyses of Anti - CTLA4 Antibody Stability Using different buffers, the binding activities of antibodies BA - 087 - 05 - 19 and BA - 087 - 08 - 32 to human CTLA4 at pH 6.0 in buffer and at pH 7.4 in buffer were measured. Both ELISA and FACS analyses were used to measure the binding activities. In the ELISA analysis, serially diluted BA - 087 - 05 - 19 and BA - 087 - 08 - 32 samples were added to wells of each buffer pre - coated with human CTLA4. The amount of bound antibody was quantified using an anti - human IgG antibody conjugated to HRP. The absorbance at 450 nm in each measurement was proportional to the amount of bound antibody. See the ELISA data in Figures 9A - 9F. The EC 50 value (ng / mL) of binding to human CTLA4 was determined by the absorbance at 450 nm against the antibody concentration using a Prism variable slope four - parameter dose - response curve, which was calculated using a non - linear fitting (variable slope, four parameters) model incorporated in GraphPad Prism software (version 7.03). Tables 10 - 11 show the EC 50 values of binding to human CTLA4 measured by ELISA in different buffers. The buffers tested included His buffer (His), Tris buffer (Tris), Glutamine buffer (Glu), and no buffer.

Table 10

Table 11

[0341] In FACS analysis, serially diluted BA-087-05-19 and BA-087-08-32 samples were added to cells expressing human CTLA4. The amount of bound antibody was quantified using an anti-human IgG antibody conjugated to a fluorophore. The MFI in each reaction was proportional to the amount of bound antibody. The binding activities measured by FACS are shown in FIGS. 10A-10F. The EC 50 value (ng / mL) for binding to human CTLA4 on cells was determined by the MFI of the singlet population against antibody concentration using a Prism variable slope four-parameter dose-response curve, which was calculated using a non-linear fitting (variable slope, four parameters) model incorporated in GraphPad Prism software (version 7.03). The EC 50 values for binding to human CTLA4 on CHO cells are shown in Tables 12-13. [Table 12] [Table 13]

[0342] Example 7: In Silico Immunogenicity Analysis of BA-087-05-19 This study determined the potential immunogenicity of BA-087-05-19 using the EpiVax In Silico Immunogenicity Screening Toolkit. The software was accessed via ISPRI, a web-based interactive screening and protein redesign interface. Using the BA-087-05-19 variable domain as input, the software was used to evaluate potential immunogenicity on a normalized scale and predict potential ADA responses.

[0343] The potential immunogenicity of BA-087-05-19 was analyzed and compared to known antibodies on a normalized scale. The data indicate that BA-087-05-19 has a Trepitope-adjusted EpiMatrix protein score of 27.70 and a predicted T-dependent antibody response of 1.29%. This predicted low immunogenicity falls within the group of optimal antibodies (having low effector and high Trepitope content).

[0344] Example 8: Functional assay of enhanced IL-2 secretion by anti-CTLA4 antibodies In this example, the functional activities of antibodies BA-087-05-19 and BA-087-08-32 in the induction of IL-2 secretion by human lymphocytes stimulated with Staphylococcus enterotoxin B (SEB) were determined. Serial dilutions of BA-087-05-19, BA-087-08-32, ipilimumab, and ipilimumab analog were added to human peripheral blood mononuclear cells (PBMC) from normal healthy donors stimulated with SEB. The ability of the antibodies to enhance IL-2 secretion in SEB-stimulated human PBMC was quantified using an IL-2 ELISA kit.

[0345] A total of three independent experiments were performed. As shown in Figure 11A, in SEB-stimulated peripheral blood mononuclear cell (PBMC) cultures, the addition of BA-087-05-19 and BA-087-08-32 enhanced IL-2 production more than the addition of isotype controls at the levels observed with ipilimumab and ipilimumab analog at pH 6.2. On the other hand, as shown in Figure 11B, IL-2 production did not increase with the addition of BA-087-05-19 and BA-087-08-32 at pH 7.4.

[0346] At a concentration of 10 μg / mL, BA-087-05-19 promoted an average 1.4-fold increase, and BA-087-08-032 promoted an average 1.5-fold increase in IL-2 production, similar to the increases observed with ipilimumab and ipilimumab analogs compared to the isotype control at pH 6.2. These results indicate that the functional activities of BA-087-05-19 and BA-087-08-32 are equivalent to the activities observed with ipilimumab and ipilimumab analogs at pH 6.2.

[0347] Example 9: Promega® CTLA4 Blocking Assay for Anti-CTLA4 Antibodies The activities of antibodies BA-087-05-19 and BA-087-08-32 in blocking the interaction between human CTLA4 and its ligands (CD80 and CD87) were determined by using an in vitro Promega® CTLA4 blocking assay. Serial dilutions of BA-087-05-19, BA-087-08-32, ipilimumab and ipilimumab analogs were added to Jurkat effector cells, followed by addition of aAPC / Raji cells according to the vendor's protocol. Blocking of the interaction between CTLA4 and its ligands results in activation of the IL-2 pathway modified in Jurkat effector cells, which was quantified using a Bio-Glo® luciferase assay kit.

[0348] As shown in Fig. 12A, the results showed that BA-087-05-19 and BA-087-08-32 were able to block the interaction between CTLA4 and its ligands (CD80 / CD87) at similar levels as observed with ipilimumab and ipilimumab analogs at pH 6.0. In contrast, as shown in Fig. 12B, BA-087-05-19 and BA-087-08-32 were less effective in interfering with the interaction between CTLA4 and its ligand at pH 7.4. These results indicate that the in vitro functional activities of BA-087-05-19 and BA-087-08-32 are equivalent to the activities observed with ipilimumab and ipilimumab analogs at pH 6.0 and are significantly lower than the activities of ipilimumab and ipilimumab at pH 7.4 when blocking.

[0349] Example 10: FACS Assay for Ligand Blockade by Anti-CTLA4 Antibodies The activities of BA-087-05-19 and BA-087-08-32 to inhibit the interaction between human CTLA4 and its ligands hB7-1 (hCD80) and hB7-2 (hCD86) were assayed by FACS, and the competitive binding of BA-087-05-19 and BA-087-08-32 at a fixed concentration to CHO cells expressing human CTLA4 in the presence of different concentrations of hB7-1 and hB7-2 was evaluated. The amounts of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4 cells were quantified using anti-human IgG antibodies conjugated to fluorophores. As shown in Figs. 13A - 13B, the mean fluorescence intensity (MFI) in each reaction was proportional to the amount of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4.

[0350] In addition, FACS analysis was used to determine the competitive binding of serially diluted BA-087-05-19 and BA-087-08-32 to CHO cells expressing human CTLA4 at fixed concentrations of hB7-1 and hB7-2. The amount of hB7-1 and hB7-2 bound to CHO-huCTLA4 cells was quantified using an anti-His antibody and an anti-mouse IgG antibody conjugated to a fluorophore. The MFI in each reaction was proportional to the amount of hB7-1 and hB7-2 bound to CHO-huCTLA4. The data showed that BA-087-05-19 and BA-087-08-32 blocked the interaction between huCTLA4 and its ligands hB7-1 and hB7-2 at levels similar to those achieved by ipilimumab and ipilimumab analogs (Figures 14A - 14B).

[0351] The data show that BA-087-05-19 and BA-087-08-32 can efficiently block the interaction between human CTLA4 and its ligands hB7-1 (hCD80) and hB7-2 (hCD86), similar to ipilimumab and ipilimumab analogs. Competitive FACS analysis was performed only at pH 6.0 because binding at pH 7.4 was very limited for BA-087-05-19 and BA-087-08-32.

[0352] Methods used in the examples ELISA assays were performed using the following protocol: 1) Coat ELISA plates with 0.5 μg / mL (06_20_17 and 06_28_17 experiments) or 1 μg / mL (07_06_17 and 07_11_17 experiments) of recombinant CTLA4 antigen in 100 μL of carbonate-bicarbonate coating buffer. 2) Cover the plates with sealing film and incubate overnight at 4°C. 3) Decant the plates and gently tap on stacked paper towels to remove any remaining liquid. 4) Wash the wells twice by dispensing 200 μL of various pH incubation buffers into the wells according to the sample map, and aspirate the contents completely. 5) Add 200 μL of various pH incubation buffers to the wells according to the sample map. Cover the plate with a sealing film and place it on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and gently tap it on a stacked paper towel to remove the remaining liquid. 7) Serial dilute the test substance in various pH incubation buffers to 250 ng / mL, 100 ng / mL, or 25 ng / mL. 8) Add 100 μL / well of the diluted test substance to the plate according to the sample map. 9) Cover the plate with a sealing film and place it on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and gently tap it on a stacked paper towel to remove the remaining liquid. 11) Wash the wells three times by dispensing 200 μL of various pH washing buffers into the wells according to the sample map and aspirating the contents completely. 12) Dilute the HRP secondary antibody 1:2500 in various pH incubation buffers 13) Add 100 μL of the HRP secondary antibody diluted in various pH incubation buffers to each well according to the sample map. 14) Cover the plate with a sealing film and place it on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and gently tap it on a stacked paper towel to remove the remaining liquid. 16) Wash the wells three times by dispensing 200 μL of various pH washing buffers into the wells according to the sample map and aspirating the contents completely. 17) Dispense 50 μL of TMB substrate solution per well into all wells of the plate. Incubate at room temperature for 3 minutes. 18) Add 50 μL of 1 N HCl per well to all wells of the plate. Read the plate at 450 nm using a Molecular Device SpectraMax 190 microplate reader. 19) Measure the raw OD450nm data. 20) Using Softmax Pro software (Molecular Devices), the average OD values (from two replicates) at different pHs were plotted against the pH of the buffer. Curve fitting was performed using a 4-parameter model incorporated in the software. The inflection point of the pH curve (50% binding activity) is equal to parameter C of the fitting equation. The binding activity at pH 6.0 was set to 100%. The pH for 90% binding activity was interpolated from the fitted curve using the "InterpX" function of Softmax Pro software.

[0353] The following protocol was used to perform a surface plasmon resonance (SPR) assay: The SPR2 / 4 instrument, SPR affinity sensor (Amine Flat), and immobilization buffer kit were manufactured by Sirra Sensors. The SPR sensor has four flow cells (FC1 - FC4) that can be addressed individually or in groups. The extracellular domain of CTLA4 was immobilized on FC2 and FC4, while BSA was immobilized on FC1 and FC3 (control surfaces).

[0354] Immobilization was carried out according to the protocol recommended by the vendor. (1) The activator was prepared by mixing 200 mM EDC and 50 mM NHS (Sierra Sensors) immediately before injection. The amine sensor chip was activated with the mixture at a flow rate of 25 μL / min for 480 seconds. (2) 25 μg / mL of human CTLA4 in 10 mM NaAc (pH 5.0) was injected into FC2 and FC4 respectively at a flow rate of 25 μL / min for 480 seconds. The chip surface was inactivated with 1 M ethanolamine-HCl (Sierra Sensors) at a flow rate of 25 μL / min for 480 seconds through FC1 - 4. (3) Under the same conditions, but without injecting the protein, the control surface was activated and inactivated. (4) Before injecting the analyte, the electrophoresis buffer was switched to PBST at the required pH. Before injecting the first analyte, the measuring instrument was equilibrated with the electrophoresis buffer for 1 hour. (5) All analyte injections were carried out at 25 μL / min and 25 °C.

[0355] BA - 087 - 05 - 19 was diluted to 5 μg / mL (34.25 nM), 2 μg / mL (13.70 nM), 1 μg / mL (6.85 nM), 0.5 μg / mL (3.42 nM), 0.2 μg / mL (1.37 nM), and 0 μg / mL (0.0 nM) in electrophoresis buffer (PBST buffer, pH 6.0 or 7.4). BA - 087 - 08 - 32 was diluted to 5 μg / mL (34.25 nM), 2 μg / mL (13.70 nM), 1 μg / mL (6.85 nM), 0.5 μg / mL (3.42 nM), 0.2 μg / mL (1.37 nM), and 0 μg / mL (0.0 nM) in electrophoresis buffer (PBST buffer, pH 6.0 or 7.4).

[0356] 100 μL of the diluted analyte BA - 087 - 05 - 19 or BA - 087 - 08 - 32 was injected onto flow cells 1 and 2 (or 3 and 4) at a flow rate of 25 μL / min for a 240 - second association period, followed by a 360 - second dissociation period. While increasing the concentration of the analyte, the electrophoresis of the analyte was repeated for 6 cycles. After each cycle of the interaction analysis, the chip surface was regenerated by injecting 6 μL of 10 mM glycine (pH 2.0). Each set was electrophoresed 3 times in total at the same pH.

[0357] A flow cell 1 (or 3) without immobilized protein was used as a control surface for reference subtraction. Furthermore, data with only buffer as the analyte (0 nM analyte) were subtracted from each electrophoresis. Using the provided analysis software Analyzer R2 (Sierra Sensors), a 1:1 binding model was used to fit the doubly subtracted data. The molar concentration of the analyte was calculated using a molecular weight of 146 kDa.

[0358] A fluorescence-activated cell sorting (FACS) assay was performed using the following protocol.

[0359] Cell staining to determine surface expression of human CTLA4 or cynomolgus monkey CTLA4 1) Seed 3×10 6 cells into a T-75 flask and culture medium according to the vendor's instructions. 2) On the day of FACS analysis, remove and discard the culture medium. 3) Briefly rinse the cell layer with PBS solution. 4) Add 1.5 mL of Detachin solution to each of the T-75 flasks. Wait until the cell layer is dispersed. 5) Add 4.5 mL of culture medium to the corresponding cell line and resuspend the cells by gentle pipetting. 6) Pool the cells and transfer the cell suspension to a 50 mL conical tube. 7) After counting the cells by trypan blue staining, centrifuge at 1500 rpm for 5 minutes at 4°C. 8) Wash the cells once with PBS and transfer 3×10 5 cells to an Eppendorf tube. 9) Add 2 μL of mouse anti-CTLA4 (mouse IgG1 conjugated with PE) or PE isotype mouse IgG1 to 100 μL of PBS solution containing 1% BSA per tube and shake at 100 RPM for 1 hour on ice. 10) Wash the cells 3 times with 150 μL of PBS solution. 11) Fix the cells with 4% PFA for 10 minutes at room temperature and wash the cells once with PBS. 12) Resuspend the cells in 100 μL of PBS and analyze the cells using a NovoCyte flow cytometer.

[0360] FACS analysis of CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4 using test antibodies. 1) Harvest the cells (as in 3.3, steps 1 - 7) and wash the cells once with PBS. 2) Resuspend the cells at 3×10 6 cells / mL in FACS buffer at pH 6.0 or pH 7.4. 3) Aliquot 100 μL of 3×10 5 cells in FACS buffer at pH 6.0 or pH 7.4 into a 96 - well U - bottom plate. 4) Centrifuge the cells and discard the buffer. 5) Serially dilute the test substance starting at 10 μg / mL in FACS buffer at pH 6.0 or pH 7.4 (for the 06 - 16 - 17, 06 - 26 - 17, and 06 - 28 - 17 experiments for a total of 8 data points), or 100 μg / mL (for the 07_10_17 experiment for a total of 11 data points) in 3 - fold dilutions. 6) Add 100 μL / well of the diluted test substance to the cells, gently mix the wells, and incubate on ice for 1 hour with shaking (100 rpm). 7) Centrifuge the cells at 1500 rpm for 5 minutes at 4°C. Wash the cells twice with 150 μL of wash buffer at pH 6.0 or pH 7.4. 8) Dilute the goat anti - human IgG AF488 antibody 1:300 in FACS buffer at pH 6.0 or pH 7.4. 9) Add 100 μL of the diluted antibody (from the above step) to the cells and incubate on ice for 45 minutes while protecting from light. 10) Pellet the cells and wash the cells three times with 150 μL of wash buffer at pH 6.0 or pH 7.4. 11) Fix the cells with 4% PFA diluted in 1X PBS for 10 minutes at room temperature and then wash the cells with 1X PBS. 12) Resuspend the cells in 100 μL of 1X PBS. 13) Analyze the cells using an Ex488nm / Em530nm with a NovoCyte flow cytometer. Collect at least 20,000 cells.

[0361] Analyze the FACS data using a non-linear fitting (variable slope, 4-parameter) model incorporated in GraphPad Prism software (version 7.03).

[0362] PROMEGA® CTLA4 Blocking Assay 1) Transfer a vial of thaw-and-use CTLA4 Jurkat effector cells (CS186912) from liquid nitrogen storage to dry ice on the bench. Thaw the vial in a 37 °C water bath until the cells are just thawed (about 2 minutes). During thawing, gently agitate while visually inspecting (do not invert). 2) Pipette the cell suspension in the vial up and down 2 - 3 times to mix gently, and transfer 0.8 mL to a tube labeled "CTLA4 cells" containing 3.2 mL of RPMI + 10% FBS. 3) Centrifuge the cells at 1500 rpm for 10 minutes and resuspend in 1 mL of RPMI + 10% FBS. Mix well, divide the cell suspension into two tubes, centrifuge the cells, wash the pellet once with either assay medium at pH 6.0 or pH 7.4, and then resuspend the cell pellet in 2 mL of assay medium at pH 6.0 or pH 7.4. 4) Immediately dispense 25 μL of CTLA4 Jurkat effector cells into the inner 60 wells of a 96-well plate according to the layout. 5) Add 100 μL of sterile water per well to the unused wells around the sample wells. 6) Starting from 300 μg / mL, perform a two-fold serial dilution of the test substance stock in pH 6.0 or pH 7.4 assay medium to generate 10-fold dilution data points. 7) Pipette 25 μL of the serially diluted 3-fold concentrated sample material stock according to the layout into wells containing 25 μL of CTLA4 Jurkat effector cells. 8) Transfer the vial of thawable CTLA4 aAPC / Raji cells (CS186911) from liquid nitrogen storage to dry ice on the bench. Thaw the vial in a 37 °C water bath until the cells are just thawed (about 2 minutes). During thawing, gently agitate while visually inspecting (do not invert). 9) Pipette the cell suspension in the vial up and down 2 - 3 times to mix gently and transfer 0.8 mL to a tube labeled "aAPC / Raji cells" containing 7.2 mL of RPMI + 10% FBS. 10) Centrifuge the cells at 1500 rpm for 10 minutes and resuspend in 1 mL of RPMI + 10% FBS. Mix well, divide the cell suspension into two tubes, centrifuge the cells, wash the pellet once with assay medium at pH 6.0 or pH 7.4, and then resuspend the cell pellet in 4 mL of assay medium at pH 6.0 or pH 7.4. 11) Immediately pipette 25 μL of CTLA4 aAPC / Raji cells into the inner 60 wells of an assay plate that already contains 50 μL of cell and antibody solution. The assay volume is 75 μL in total. 12) Place a lid on the plate and incubate the plate at 37 °C for 16 hours in a 5% CO2 humidified incubator. 13) During the 16-hour induction period, warm the Bio-Glo™ buffer to ambient temperature using a room temperature water bath before adding it to the Bio-Glo™ substrate. 14) Reconstitute the Bio-Glo™ luciferase assay system by transferring 1 bottle of Bio-Glo™ buffer to the bottle containing the Bio-Glo™ substrate. 15) After 16 hours of induction, remove the assay plate from the CO2 incubator and equilibrate at ambient temperature for 15 minutes. 16) Add 75 μL of Bio-Glo™ reagent to the inner 60 wells of the assay plate. 17) Incubate the plate at ambient temperature for 5 - 10 minutes. 18) Record the luminescence using a SpectraMax i3X plate reader.

[0363] Along with the details of the structure and function of the present invention, many features and advantages of the present invention are described in the foregoing description. However, it should be understood that the present disclosure is merely exemplary, and that detailed changes may be made in matters regarding the shape, size, and arrangement of components within the principles of the present invention, within the full scope indicated by the broad general meaning of the terms in which the appended claims are expressed.

[0364] All documents referred to herein are incorporated herein by reference in their entirety, or they provide a particularly reliable disclosure. Applicant(s) do not intend to dedicate the disclosed embodiments to the public, and unless the disclosed modifications or changes are clearly within the scope of the claims, they are considered to be part of the present invention under the doctrine of equivalents.

[0365] SEQUENCE LISTING <110> BIOATLA LLC <120> Anti-CTLA4 antibodies, antibody fragments and their immunoconjugates and uses thereof <130> BIAT-1028WO <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 1 Gly Phe Thr Phe Ser His Tyr Thr Met His 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic sequence <220> <221> Variants <222> (3)..(3) <223> This may be amino acid S or D <220> <221> Variants <222> (5)..(5) <223> This may be amino acid D, H or I <220> <221> Variants <222> (8)..(8) <223> This may be amino acid N or Y <220> <221> Variants <222> (10)..(10) <223> This may be amino acid Y or I <220> <221> Variants <222> (11)..(11) <223> This may be amino acid Y or E <220> <221> Variants <222> (13)..(13) <223> This may be amino acid D or K <220> <221> Variants <222> (15)..(15) <223> This may be amino acid V or M <400> 2 Phe Ile Xaa Tyr Xaa Gly Asn Xaa Lys Xaa Xaa Ala Xaa Ser Xaa Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic sequence <220> <221> Variants <222> (9)..(9) <223> This amino acid may be Y or I <400> 3 Thr Gly Trp Leu Gly Pro Phe Asp Xaa 1 5 <210> 4 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic sequence <220> <221> Variants <222> (2)..(2) <223> This amino acid may be A or I <220> <221> Variants <222> (5)..(5) <223> This amino acid may be Y, S or H <220> <221> Variants <222> (6)..(6) <223> This amino acid may be V or G <400> 4 Arg Xaa Ser Gln Xaa Xaa Gly Ser Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic sequence <220> <221> Variants <222> (9)..(9) <223> This amino acid may be V or I <400> 5 Gly Ala Phe Ser Arg Ala Thr Gly Xaa 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 6 Gln Gln Asp Gly Ser Ser Pro Trp Thr 1 5 <210> 7 <211> 108 <212> PRT <213> artificial <220> <223> synthetic sequence <400> 7 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 9 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 9 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 11 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 11 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Phe Ile Ser Tyr His Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 13 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 13 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 14 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Phe Ile Asp Tyr His Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 15 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 15 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Gly Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 16 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Asn Lys Ile Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 17 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Gly Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Asn Lys Ile Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 19 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 19 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 20 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 20 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Tyr His Gly Asn Asn Lys Tyr Glu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 21 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 21 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 22 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Tyr His Gly Asn Asn Lys Tyr Glu Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 23 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 23 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 24 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 25 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 25 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 26 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 27 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 27 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 28 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 28 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 29 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 29 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 30 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Asp Gly Asn Tyr Lys Tyr Tyr Ala Lys Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 31 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 31 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Ile Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 33 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 33 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 34 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 34 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Ile Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 35 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 35 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 36 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 36 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Ile Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 37 <211> 108 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 37 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln His Val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 38 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Thr Met His Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45 Gly Phe Ile Ser Tyr Ile Gly Asn Tyr Lys Tyr Tyr Ala Asp Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 39 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 39 Gly Phe Thr Phe Ser His Tyr Thr Met His 1 5 10 <210> 40 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 40 Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 41 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 41 Thr Gly Trp Leu Gly Pro Phe Asp Tyr 1 5 <210> 42 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 42 Arg Ala Ser Gln Tyr Val Gly Ser Ser Tyr Leu Ala 1 5 10 <210> 43 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 43 Gly Ala Phe Ser Arg Ala Thr Gly Ile 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 44 Gln Gln Asp Gly Ser Ser Pro Trp Thr 1 5

Claims

1. An isolated polypeptide that specifically binds to CTLA4 protein, wherein said polypeptide comprises a heavy chain variable region comprising three complementarity determining regions, said regions having H1, H2, and H3 sequences, (a) said H1 sequence is GFTFSHYTMH (SEQ ID NO: 1), (b) the H2 sequence is FIX １ YX ２ GNX ３ KX ４ X ５ AX ６ SX ７ KG (SEQ ID NO: 2), and (c) the H3 sequence is TGWLGPFDX ８ (SEQ ID NO: 3), and In the formula, X １ is S or D, and X ２ is D, H or I, and X ３ is N or Y, and X ４ is Y or I, and X ５ is Y or E, and X ６ is D or K, and X ７ is V or M, and X ８ is Y or I, a polypeptide.

2. The polypeptide according to claim 1, wherein said H2 sequence is selected from FIDYHGNNKYYYADSVKG, FISYDGNNKIYYADSVKG, FISYDGNNKYYYADSVKG, FISYDGNYKYYYADSVKG, FISYDGNYKYYAKSVKG, FISYHGNNKYEADSVKG, FISYHGNNKYYYADSVKG, FISYIGNYKYYADSMPKG, FISYIGNYKYYYADSVKG.

3. The polypeptide according to any one of claims 1 to 2, wherein said H3 sequence is selected from TGWLGPFDY and TGWLGPFDI.

4. The polypeptide according to claim 1, wherein said heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38.

5. The polypeptide according to any one of claims 1 to 4, combined with an isolated light chain variable region comprising three complementarity determining regions having L1, L2, and L3 sequences, (a) the L1 sequence is RX ９ SQX １０ X １１ GSSYLA (SEQ ID NO: 4), and (b) the L2 sequence is GAFSRATGX １２ (SEQ ID NO: 5), and (c) said L3 sequence is QQDGSSPWT (SEQ ID NO: 6), In the formula, X ９ is A or I, X １０ is Y, S or H, X １１ is V or G, X １２ is V or I, a polypeptide.

6. The polypeptide according to claim 5, wherein said L1 sequence is selected from RASQHVGSSYLA, RASQSVGSSYLA, RASQYGGSSYLA, RASQYVGSSYLA, and RISQYVGSSYLA.

7. The polypeptide according to claim 6, wherein said L2 sequence is selected from GAFSRATGI and GAFSRATGV.

8. The polypeptide according to claim 5, wherein said light chain variable region has an amino acid sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37.

9. An isolated polypeptide that specifically binds to CTLA4 protein, wherein said polypeptide comprises a light chain variable region comprising three complementarity determining regions having L1, L2, and L3 sequences, (a) the L1 sequence is RX ９ SQX １０ X １１ GSSYLA (SEQ ID NO: 4), and (b) the L2 sequence is GAFSRATGX １２ (SEQ ID NO: 5), and (c) said L3 sequence is QQDGSSPWT (SEQ ID NO: 6), wherein, X ９ is A or I, X １０ is Y, S or H, X １１ is V or G, X １２ is V or I, a polypeptide.

10. The polypeptide according to claim 9, wherein the L1 sequence is selected from RASQHVGSSYLA, RASQSVSGSSYLA, RASQYGGSSYLA, RASQYVGSSSYLA, and RISQYVGSSSYLA.

11. The polypeptid according to claim 10, wherein the L2 sequence is selected from GAFSRATGI and GAFSRATGV.

12. The polypeptide according to claim 9, wherein the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37.

13. An anti-CTLA4 antibody or antibody fragment comprising the isolated polypeptide according to any one of claims 1 to 4.

14. The antibody or antibody fragment according to claim 13, further comprising the isolated polypeptide according to any one of claims 9 to 12.

15. The antibody or antibody fragment according to any one of claims 13 to 14, wherein the antibody or antibody fragment has a higher binding affinity for CTLA4 protein at the value of the condition in the tumor microenvironment compared to the different value of the same condition occurring in the non-tumor microenvironment.

16. The antibody or antibody fragment according to claim 15, wherein the condition is pH.

17. The antibody or antibody fragment according to claim 16, wherein the pH in the tumor microenvironment ranges from 5.0 to 6.8 and the pH in the non-tumor microenvironment ranges from 7.0 to 7.

6.

18. The antibody or antibody fragment according to any one of claims 13 to 17, wherein the antibody or antibody fragment has a ratio of the binding affinity for CTLA4 protein at the value of the condition in the tumor microenvironment to the binding affinity for CTLA4 protein at the different value of the same condition in the non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:

1.

19. The antibody or antibody fragment according to any one of claims 13 to 18, wherein the antibody or antibody fragment is a chimeric antibody, a multispecific antibody, or a humanized antibody.

20. An immunoconjugate comprising the antibody or antibody fragment according to any one of claims 13 to 19.

21. The immunoconjugate according to claim 20, wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cell division inhibitor, and a cytotoxic agent.

22. The immunoconjugate according to claim 21, comprising at least two of said agents.

23. The immunoconjugate according to any one of claims 21 to 22, wherein the antibody or antibody fragment and the at least one agent are covalently bound to a linker molecule.

24. The immunoconjugate according to any one of claims 21 to 23, wherein the at least one agent is selected from maytansinoids, auristatins, dolastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

25. A polypeptide according to any one of claims 1 to 12, an antibody or antibody fragment according to any one of claims 13 to 19, or an immunoconjugate according to any one of claims 20 to 24, and a pharmaceutically acceptable carrier, a pharmaceutical composition.

26. The pharmaceutical composition according to claim 25, further comprising an isotonicity agent.

27. A single-dose pharmaceutical composition according to any one of claims 25 to 26, comprising an amount of about 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg of a polypeptide according to any one of claims 1 to 12, an antibody or antibody fragment according to any one of claims 13 to 19, or an immunoconjugate according to any one of claims 20 to 24.

28. A single-dose pharmaceutical composition according to any one of claims 25 to 26, comprising an amount in the range of 135 mg to 235 mg, 235 mg to 335 mg, 335 mg to 435 mg, 435 mg to 535 mg, 535 mg to 635 mg, 635 mg to 735 mg, 735 mg to 835 mg, 835 mg to 935 mg, 935 mg to 1035 mg, 1035 mg to 1135 mg, 1135 mg to 1235 mg, or 1235 mg to 1387 mg of the polypeptide according to any one of claims 1 to 12, the antibody or antibody fragment according to any one of claims 13 to 19, or the immunoconjugate according to any one of claims 20 to 24.

29. A pharmaceutical composition according to any one of claims 25 to 28, further comprising an immune checkpoint inhibitor molecule different from the polypeptide according to any one of claims 1 to 12 and the antibody or antibody fragment according to any one of claims 13 to 19.

30. The pharmaceutical composition according to claim 29, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.

31. The pharmaceutical composition according to claim 30, wherein the immune checkpoint is selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS.

32. The pharmaceutical composition according to claim 30, wherein the immune checkpoint is PD-1 or PD-L1.

33. A pharmaceutical composition according to any one of claims 25 to 32, further comprising an antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT protein.

34. The pharmaceutical composition according to claim 33, wherein the WNT protein is selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

35. A method for treating cancer, comprising the step of administering to a patient having cancer a polypeptide according to any one of claims 1 to 12, an antibody or antibody fragment according to any one of claims 13 to 19, an immunoconjugate according to any one of claims 20 to 24, or a pharmaceutical composition according to any one of claims 25 to 34.

36. A kit for diagnosis or treatment, the kit comprising a polypeptide according to any one of claims 1 to 12, an antibody or antibody fragment according to any one of claims 13 to 19, an immunoconjugate according to any one of claims 20 to 24, or a pharmaceutical composition according to any one of claims 25 to 34, and instructions for using the antibody or antibody fragment, the immunoconjugate, and / or the pharmaceutical composition for diagnosis or treatment.

37. An anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 39 to 41, and the light chain variable region comprises three complementarity-determining regions having the amino acid sequences of SEQ ID NOs: 42 to 44.

38. The antibody according to claim 37, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 8 and the light chain variable region has the amino acid sequence of SEQ ID NO:

7.

39. The antibody according to any one of claims 37 to 38, wherein the antibody has a higher binding affinity for CTLA4 protein at a value of a condition in the tumor microenvironment as compared to a different value of the same condition occurring in the non-tumor microenvironment.

40. The antibody according to claim 39, wherein the condition is pH.

41. The antibody according to claim 40, wherein the pH in the tumor microenvironment ranges from 5.0 to 6.8 and the pH in the non-tumor microenvironment ranges from 7.0 to 7.

6.

42. The antibody according to any one of claims 37 to 41, having a ratio of the binding affinity of the antibody to the CTLA4 protein at a value of a condition in the tumor microenvironment to the binding affinity of the antibody to the CTLA4 protein at a different value of the same condition in the non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:

1.

43. The antibody according to any one of claims 37 to 42, wherein the antibody is a chimeric antibody or a humanized antibody.

44. An immunoconjugate comprising the antibody according to any one of claims 37 to 43.

45. The immunoconjugate according to claim 44, wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cell division inhibitor, and a cytotoxic agent.

46. The immunoconjugate according to claim 45, comprising at least two of said agents.

47. The immunoconjugate according to any one of claims 45 to 46, wherein the antibody and the at least one agent are covalently linked to a linker molecule.

48. The immunoconjugate according to any one of claims 46 to 47, wherein the at least one agent is selected from maytansinoids, auristatins, dolastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

49. The antibody according to any one of claims 37 to 43, or the immunoconjugate according to any one of claims 44 to 48, and A pharmaceutically acceptable carrier, a pharmaceutical composition.

50. The pharmaceutical composition according to claim 49, further comprising an isotonicity agent.

51. A method of treating cancer, comprising administering to a patient having cancer the antibody according to any one of claims 37 to 43, or the immunoconjugate according to any one of claims 44 to 48, or the pharmaceutical composition according to any one of claims 49 to 50.

52. A kit for diagnosis or treatment, comprising the antibody according to any one of claims 37 to 43, the immunoconjugate according to any one of claims 44 to 48, or the pharmaceutical composition according to any one of claims 49 to 50, and an instruction manual for using the antibody, the immunoconjugate, and / or the pharmaceutical composition for diagnosis or treatment.

Images