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

Abstract

To provide a pharmaceutical composition containing anti CTLA-4 antibody for treating cancer.SOLUTION: There are provided a polypeptide having a heavy chain variable region and / or a light chain variable region which specifically bind to a CTLA4 protein, and an antibody and antibody fragment including a heavy chain variable region and / or a light chain variable region which bind to the CTLA4 protein. Further, also provided are a pharmaceutical composition and a kit which include the polypeptide, or an antibody and an antibody fragment including the polypeptide. The pharmaceutical composition is for treating a patient having small cell lung cancer, non-small cell lung cancer, kidney cancer, liver cancer, melanoma, bladder cancer, cervical cancer, hepatocellular carcinoma, stomach cancer or kidney cancer.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, as well as the use of the antibodies, antibody fragments, and immunoconjugates in methods of diagnosis and treatment. [Background technology]

[0002] The vertebrate immune system requires multiple signals to achieve optimal immune activation. See, e.g., Janeway, Cold Spring Harbor Symp. Ouant. Biol. 54:1-14 (1989); Paul William E., ed. Raven Press, NY, Fundamental Immunology, 4th edition (1998), especially pages 12, 13, and 411-478. Interactions between T lymphocytes (T cells) and antigen-presenting cells (APCs) are essential for immune activation. The levels of many of the aggregation 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)). Increased levels of these molecules may help explain why activated APCs are more effective than resting APCs at stimulating antigen-specific proliferation of T cells (Kaiuchi et al., J. Immunol., 131:109-114 (1983); Kreiger 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] The T cell immune response is a complex process that involves cell-cell interactions, particularly between T cells and accessory cells such as APCs (Springer et al., A. Rev. Immunol., 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 a number of 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 monoclonal antibody reactivity on the surface of the cell (McMichael, Ed., Leukocyte Tipping III, Oxford Univ. Press, Oxford, NY (1987)).

[0004] CTLA4 is a T cell surface molecule originally identified by differential screening of a cDNA library of mouse cytolytic 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 may function in cytolytic responses (Brunet et al. (see above); Brunet et al., Immunol. Rev., 103:21-36 (1988)). Researchers have reported the cloning of the gene for the human counterpart of CTLA4 and its mapping to the same chromosomal region (2d, 33-34) as CD28 (Dariavach et al., Eur: J. Immunol., 18:1901-1905 (1988)), CD28 (Lafage-Pochitaloff et al. Immunogenetics, 31:198-201 (1990)). Sequence comparison between the DNA of human CTLA4 and the DNA encoding the CD28 protein revealed significant sequence homology, with the greatest degree of homology in the juxtamembrane and cytoplasmic regions (Brunet et al., 1988 (see above); Dariavach et al., 1988 (see above)).

[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. Patent 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)). It has been reported that CTLA4-deficient mice suffer from massive lymphoproliferation (Chambers et al., supra). Blockade of CTLA4 has also been reported to enhance T cell responses in vitro (Walunas et al., Immunity, 1:405-413 (1994)) and in vivo (Kearney, J. Immunol., 155:1032-1036 (1995)), exacerbate antitumor immunity (Leach, Science, 271:1734-1736 (1996)), and enhance induced autoimmune disease (Luhder, J Exp. Med., 187:427-432 (1998)). CTLA4 has also been reported to have alternative or additional effects on early features 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. CTLA4 blocking antibodies 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 intrinsic and extrinsic 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, leading to inhibition of antigen presentation by APCs and inhibition of T cell function, (iii) binding of CTLA4 to CD80 / CD86 reduces the availability of ligand for binding by CD28, and (iv) binding of CTLA4 to CD80 / CD86 inhibits their transendocytosis. and 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 the TCR; (vi) CTLA4 acts as a high-affinity competitor to occupy CD80 / 86, thereby preventing binding by CD28; (vii) soluble splice variants of CTLA4 may be able to inhibit T cell activation; and (viii) CTLA4 inhibits T cell stop signals that are important for T cell activation by APCs.

[0007] Thus, inhibition of CTLA4 has been shown to promote adaptive immune response and stimulation of 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 have been investigated as a strategy to promote antitumor immunity in cancer. Blocking CTLA4 is also a promising therapeutic strategy for disorders associated with T cell exhaustion, such as chronic viral infections.

[0008] Antibodies against CTLA4 have been developed before. US Patent No. 9,758,583 discloses antibodies or antibody fragments that are said to bind to either or both human and mouse CTLA4, which can be formulated into compositions for the treatment of cancer. Some of the antibodies or antibody fragments are also said to optionally 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] US2009 / 0252741 also 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 the 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 are said to be capable of blocking human CTLA4 from binding to human B7, and are therefore suitable for treating diseases with inflammatory or allergic components, such as 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 with inflammatory or allergic components, such as graft-versus-host disease, host-versus-graft disease, allergies, autoimmune diseases, and other inflammatory diseases.

[0011] Although antibodies against CTLA4 are known and commercially available, it is desirable to find improved anti-CTLA4 antibodies suitable for cancer therapy with reduced or minimal side effects. The present invention provides anti-CTLA4 antibodies or antibody fragments suitable for therapeutic and diagnostic use, particularly for cancer diagnosis and treatment. 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 may provide a more selective treatment of CTLA4 in tumors, and as a result of selectivity for CTLA4 in tumors, higher doses of these anti-CTLA4 antibodies or antibody fragments may be possible, which may result in more effective therapeutic treatment without a corresponding increase in undesirable side effects. Summary of the Invention

[0012] In one aspect, the present invention provides isolated heavy chain variable region polypeptides that specifically bind to a 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), In the formula, 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 combining any of the isolated heavy chain variable region polypeptides described above with an isolated light chain variable region polypeptide selected from an isolated light chain variable region polypeptide comprising three complementarity determining regions having an L1, L2, and L3 sequence, L1 arrangement 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), In the formula, X9 is A or I, and X 10 is Y, S or H, and X 11 is V or G, and X 12 is V or I.

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

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

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

[0017] In each of the foregoing embodiments, the L2 sequence may 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 NOs:7 and 8, SEQ ID NOs:9 and 10, SEQ ID NOs:11 and 12, SEQ ID NOs:13 and 14, SEQ ID NOs:15 and 16, SEQ ID NOs:17 and 18, SEQ ID NOs:19 and 20, SEQ ID NOs:21 and 22, SEQ ID NOs:23 and 24, SEQ ID NOs:25 and 26, SEQ ID NOs:27 and 28, SEQ ID NOs:29 and 30, SEQ ID NOs:31 and 32, SEQ ID NOs:33 and 34, SEQ ID NOs:35 and 36, and SEQ ID NOs:37 and 38.

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

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

[0022] In yet another aspect, the present invention provides an immunoconjugate comprising any of the antibodies or antibody fragments of the present invention described above, in which the antibody or antibody fragment may be conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent.

[0023] In yet another aspect, the present invention provides a pharmaceutical composition comprising any of the above-described polypeptides, antibodies, antibody fragments, and immunoconjugates of the present invention together with a pharma- ceutically acceptable carrier. A single dose of the pharmaceutical composition may comprise an amount 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 of the polypeptide, antibody, antibody fragment, or immunoconjugate.

[0024] A single dose of the pharmaceutical composition may comprise 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 above pharmaceutical compositions can 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 aforementioned 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 a WNT protein. 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 diagnostic or therapeutic kit, comprising any 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, wherein the heavy chain variable region comprises three complementarity determining regions having the amino acid sequences of SEQ ID NOs: 39-41, and the light chain variable region comprises three complementarity determining regions having the amino acid sequences of SEQ ID NOs: 42-44.

[0029] In the foregoing embodiment, 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] 1 shows a sequence alignment of exemplary heavy chain variable regions of anti-CTLA4 antibodies of the invention. [Diagram 2] 1 shows a sequence alignment of an exemplary light chain variable region of an anti-CTLA4 antibody of the present invention. [Figure 3A] 1 shows a comparison of the binding activity to human CTLA4 of two of the anti-CTLA4 antibodies of the invention versus ipilimumab and an ipilimumab analog (Ipi analog) at pH 6.0 as measured by enzyme-linked immunosorbent assay (ELISA). [Figure 3B]3B shows a comparison of the binding activity to human CTLA4 of the two anti-CTLA4 antibodies of the invention of FIG. 3A versus ipilimumab and an ipilimumab analog (Ipi analog) at pH 7.4 as measured by ELISA. [Figure 4A] 3B shows a comparison of the binding activity of two anti-CTLA4 antibodies of the invention in FIG. 3A to cynomolgus monkey CTLA4 versus ipilimumab and an ipilimumab analog (Ipi analog) at pH 6.0 as measured by ELISA. [Figure 4B] 3B shows a comparison of the binding activity of two anti-CTLA4 antibodies of the invention in FIG. 3A to cynomolgus monkey CTLA4 versus ipilimumab and an ipilimumab analog (Ipi analog) at pH 7.4 as measured by ELISA. [Diagram 5] FIG. 3B shows a comparison of the pH-dependent binding activity to human CTLA4 of the two anti-CTLA4 antibodies of the invention in FIG. 3A versus ipilimumab and an ipilimumab analog (Ipi analog). [Figure 6A] FIG. 3B shows a comparison of the half maximal effective concentration (EC50) and binding activity to human CTLA4 of two anti-CTLA4 antibodies of the invention in FIG. 3A relative to ipilimumab and ipilimumab analogs (Ipi analogs) at pH 6.0 as measured by fluorescence-activated cell sorting (FACS) using CHO cells. [Figure 6B] 3B shows a comparison of the EC50 and binding activity of two anti-CTLA4 antibodies of the invention in FIG. 3A to human CTLA4 versus ipilimumab and an ipilimumab analog (Ipi analog) at pH 7.4 as measured by FACS using CHO cells. [Figure 7A] FIG. 3B shows a comparison of the EC50 and binding activity of two anti-CTLA4 antibodies of the invention in FIG. 3A to cynomolgus CTLA4 at pH 6.0, as measured by fluorescence-activated cell sorting (FACS) using CHO cells, versus ipilimumab and ipilimumab analogs (Ipi analogs). [Figure 7B]3B shows a comparison of the EC50 and binding activity of two anti-CTLA4 antibodies of the invention in FIG. 3A to cynomolgus CTLA4 versus ipilimumab and ipilimumab analogs (ipi analogs) at pH 7.4 as measured by FACS using CHO cells. [Figure 8A] 3B shows a comparison of the EC50 and saturation of human CTLA4 by the anti-CTLA4 antibodies of the invention of FIG. 3A versus ipilimumab analogs at pH 7.4 as measured by FACS. [Figure 8B] 3B shows a comparison of EC50 and saturation of cynomolgus CTLA4 by the anti-CTLA4 antibodies of the invention of FIG. 3A versus ipilimumab analogs at pH 7.4 as measured by FACS. [Figure 9A-9F] 3B shows the binding activity of the anti-CTLA4 antibodies of the invention of FIG. 3A to human CTLA4 in the presence of different buffers at pH 6.0 or pH 7.4, as well as at pH 6.0 or pH 7.4, as measured by ELISA. [Figure 10A-10F] 3B shows the binding activity of the anti-CTLA4 antibodies of the invention in FIG. 3A to human CTLA4 in the presence of different buffers at pH 6.0 or pH 7.4, as well as at pH 6.0 or pH 7.4, as measured by FACS. [Figure 11A] 3B shows a comparison of the activity of the anti-CTLA4 antibodies of the present invention in FIG. 3A versus ipilimumab, ipilimumab analogs (ipi analogs), an IgG control, and nivolumab (Nivo) analogs in blocking secretion of IL2 in peripheral blood mononuclear cell (PBMC) cultures at pH 6.2. [Figure 11B] 3B shows a comparison of the activity of the anti-CTLA4 antibodies of the invention in FIG. 3A versus ipilimumab, an ipilimumab analog (ipi analog), an IgG control, and a nivolumab analog in blocking secretion of IL2 in PBMC cultures at pH 7.4. [Figure 12A] 3B shows a comparison of the EC50 and activity of the anti-CTLA4 antibodies of the invention of FIG. 3A versus ipilimumab, an ipilimumab analog (ipi analog), and an IgG control at pH 6.0 to block the interaction between CTLA4 and its ligands. [Figure 12B] 3B shows a comparison of the EC50 and activity of the anti-CTLA4 antibodies of the invention of FIG. 3A versus ipilimumab, an ipilimumab analog (ipi analog), and an IgG control in blocking the interaction between CTLA4 and its ligands at pH 7.4. [Figure 13A] A comparison of the EC50 and activity of the anti-CTLA4 antibodies of the invention in Figure 3A versus ipilimumab, ipilimumab analogs (ipi analogs), and an IgG control in competitive binding to human CTLA4 as measured by FACS is shown as a function of the concentration of the CTLA4 ligand hB7-1 (hCD80). [Figure 13B] A comparison of the EC50 and activity of the anti-CTLA4 antibodies of the invention in Figure 3A versus ipilimumab, ipilimumab analogs (ipi analogs), and an IgG control in competitive binding to human CTLA4 as measured by FACS is shown as a function of the concentration of the CTLA4 ligand hB7-2 (hCD86). [Figure 14A] A comparison of the activity of the anti-CTLA4 antibodies of the invention in Figure 3A versus ipilimumab, ipilimumab analogs (ipi analogs), and IgG controls in competitive binding to human CTLA4 at a fixed concentration of the CTLA4 ligand hB7-1 as measured by FACS is shown as a function of antibody concentration. [Figure 14B] A comparison of the activity of the anti-CTLA4 antibodies of the invention in Figure 3A versus ipilimumab, ipilimumab analogs (ipi analogs), and IgG controls in competitive binding to human CTLA4 at a fixed concentration of the CTLA4 ligand hB7-2 as measured by FACS is shown as a function of antibody concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0032] The term "about" as used herein in connection with a measured quantity refers to normal variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising caution 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] The term "affinity" as used herein refers to the strength of the sum 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, as 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 a 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 that has one or more modifications in one or more complementarity determining regions that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0035] The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (-COOH), preferably either as a free group or alternatively after condensation as part of a peptide bond. "The 20 naturally encoded polypeptide-forming α-amino acids" are art recognized 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] The term "antibody" as used herein refers to intact immunoglobulin molecules as well as fragments of immunoglobulin molecules capable of binding to an epitope of an antigen, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments that retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived can be generated using methods well known in the art (see, e.g., Harlow and Lane, supra), and are further described as follows. Antibodies can be used to isolate preparative amounts of antigens by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or staging of disease (e.g., neoplasia), as well as therapeutic applications for treating disease (e.g., neoplasia, autoimmune disease, AIDS, cardiovascular disease, infectious disease, etc.). Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0037] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0038] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to obtain a molecule consisting of an intact light chain and a portion of the heavy chain. In this manner, two Fab' fragments are obtained per antibody molecule treated.

[0039] The (Fab')2 fragment of an antibody can be obtained by treating whole antibody molecules 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] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

[0041] The term "antibody fragment" as used herein refers to a molecule other than an intact antibody that contains a portion 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, diabodies, linear antibodies, single-chain antibody molecules (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 CTLA4" refer to an antibody that can bind CTLA4 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CTLA4. In one embodiment, the extent of binding of an anti-CTLA4 antibody to unrelated, non-CTLA4 proteins is less than about 10% of the binding of the antibody to CTLA4, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds CTLA4 has an affinity 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~10 -13 M, for example, 10 -9 M~10 -13 M) in certain embodiments, the anti-CTLA4 antibody binds to an epitope of CTLA4 that is conserved among CTLA4 from different species.

[0043] The term "binding" as used herein refers to the interaction of an antibody variable region or Fv with an antigen, with the interaction being dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a particular protein structure, rather than proteins in general. As used herein, the term "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds or associates with a particular antigen more frequently, rapidly, for a longer duration, and / or with a higher affinity than with other proteins. For example, an antibody variable region or Fv specifically binds to its antigen with a higher affinity, avidity, readily, and / or for a longer duration than it binds to other antigens. In another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with substantially higher affinity than the affinity for related proteins or other cell surface proteins or antigens commonly recognized by polyreactive natural antibodies (i.e., natural antibodies known to bind to a variety of antigens naturally found in humans). However, "specific binding" does not necessarily require exclusive or undetectable binding of another antigen, which is meant by the term "selective binding." In one example, "specific binding" of an antibody variable region or Fv (or other binding region) means that the antibody variable region or Fv binds to an 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] The terms "cancer" and "cancerous" as used herein refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancer 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 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 disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0046] The term "chemotherapeutic agent" as used herein refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins, particularly (including bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (among others) (including synthetic analogs of adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, metostatin, methadone ... nitrogen mustards, such as chlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega 11 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor;Dynemicins, including dynemicins A; esperamicins; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycins, autramycins, azaserine, bleomycins, cactinomycins, carabicins, caminomycins, carzinophilins, chromomycins, dactinomycins, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); Folic acid analogues such as terin, methotrexate, pteropterin, trimetrexate;purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal agents such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as floric acid;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;Demecolcine; Diazicon; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidamol; Nitraerin; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, anguidin); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; trexates; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas that prevent the formation of microtubules by tubulin polymerization, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMF®); retinoids such as retinoic acid, including bexarotene (TARGRETIN®);clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), bisphosphonates such as NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (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 THERATOOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH ( For example, ABARELIX®; BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; 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 pharmacologic acceptable salts, acids or derivatives of any of the above; combinations of two or more of the above, such as CHOP (an abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone);and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™);

[0047] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote the growth of cancer. They may be hormones themselves, including, but are not limited to, antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), selective estrogen receptor modulators (SERMs), such as trioxyfene, keoxifene, and SERM3; pure antiestrogens without the agonist properties of fulvestrant (FASLODEX®) and EM800 (such as agents that may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), as well as anastrazole (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 androgens / retinoids such as fluoxymesteronde, all-trans retionic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide, and bicalutamide; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[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] The term "class" of an antibody as used herein 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, some of which 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] The term "conditionally active antibody" as used herein refers to an antibody that is more active under conditions of a tumor microenvironment compared to conditions of a non-tumor microenvironment. Conditions in a tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, low oxygen, lower concentrations of glucose, and slightly higher temperatures compared to a non-tumor microenvironment. For example, a conditionally active antibody is effectively inactive at normal body temperature, but is active at higher temperatures in a tumor microenvironment. In yet another embodiment, a conditionally active antibody is less active in normal oxygenated blood, but is more active under the hypoxic environment present in a tumor. In yet another embodiment, a conditionally active antibody is less active at normal physiological pH 7.2-7.8, but is more active under acidic pH 5.8-7.0, or 6.0-6.8, present in a tumor microenvironment. Other conditions known to those skilled in the art exist in the tumor microenvironment and can also be used as conditions in the present invention under which an anti-CTLA4 antibody has a different binding affinity for CTLA4.

[0051] The term "constitutive" as used herein, for example as applied to the activity of CTLA4, refers to the sustained signaling activity of a receptor kinase that is not dependent on the presence of a ligand or other activating molecule. 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). The cellular events that lead to the activation of receptor kinases are well known to those skilled in the art. For example, activation may include oligomerization into higher order receptor complexes, e.g., dimerization, trimerization, etc. The complex may comprise a single type of protein, i.e., a homomeric complex. Alternatively, the complex may comprise at least two different types of proteins, i.e., a heteromeric complex. Complex formation may be caused, for example, by overexpression of normal or mutant forms of the receptor on the surface of the cell. Complex formation may also be caused by specific mutations or mutations in the receptor.

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

[0053] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell 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 radioactive isotopes of Lu), chemotherapeutic agents or 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 nucleases 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, as well as various anti-tumor or anti-cancer agents disclosed below.

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

[0055] The term "detectably labeled" as used herein refers to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, indicates the presence of CTCs in a sample. Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that are directly or indirectly detectable based on light absorption, 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 group of molecules that are indirectly detectable based on light absorption or fluorescence, include various enzymes that convert suitable substrates, for example, from non-light absorbing molecules to light absorbing molecules, or from non-fluorescent molecules to fluorescent molecules.

[0056] The term "diagnosis" as used herein refers to determining a subject's susceptibility to a disease or disorder, determining whether a subject currently suffers from a disease or disorder, prognosis of a subject suffering from a disease or disorder (e.g., identifying a pre-metastatic or metastatic cancer state, the stage of a cancer, or the responsiveness of a cancer to treatment), and treatment (e.g., monitoring a subject's condition to provide information regarding the efficacy or effectiveness of treatment). In some embodiments, the diagnostic methods of the present invention are particularly useful in detecting early cancer.

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

[0058] The term "effector function" as used herein refers to the biological activities attributable to the Fc region of an antibody, which 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 an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, 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 native sequence Fc regions and variant Fc regions. In one embodiment, a 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 is according to the EU numbering system (also called 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] The term "framework" or "FR" as used herein refers to residues of a variable domain other than those of the hypervariable region (HVR or H1-3 in the heavy chain, L1-3 in the light chain). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences are H (or V L ), generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0062] The terms "full-length antibody," "intact antibody," or "whole antibody" refer to the antigen-binding variable region (V H or V L), as well as a light chain constant domain (CL) and a heavy chain constant domain (CH1, CH2, and CH3). The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domains of their heavy chains, full-length antibodies can be assigned to different "classes". There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the 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 cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

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

[0065] As used herein, a "human consensus framework" refers to a human immunoglobulin V L or V HIn selecting framework sequences, the framework is chosen to represent the most commonly occurring amino acid residues. L or V H The sequences are selected from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as 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, the V L With respect to V, the subgroup is subgroup κI as in Kabat et al. (see above). H With respect to the subgroup, it is subgroup III as in Kabat et al. (see above).

[0066] The term "humanized" antibody as used herein refers to a chimeric antibody that comprises amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0067] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs, with the V H There are three (H1, H2, H3) and V LThere are three HVRs (L1, L2, L3). HVRs generally include amino acid residues from the hypervariable loops and / or "complementarity determining regions" (CDRs), the latter of which are most variable in sequence and / or 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, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2, and amino acid residues 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). H Except for CDR1 of the present invention, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also comprise "specificity determining residues" or "SDRs", which are residues that contact the antigen. SDRs are contained within the regions of CDRs and are referred to as 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, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2, and amino acid residues 95-102 of H3 (see Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al. (see above).

[0068] As used herein, the term "immunoconjugate" is 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 radioactive atom, or a cytostatic agent.

[0069] The term "individual" or "subject" as used herein refers 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 cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, including inducing cell death.

[0071] The term "isolated" antibody as used herein is one that is separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity of more than 95% or more than 99%, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase high performance liquid chromatography (HPLC)). For a review of methods for evaluating 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 a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where 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 the antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present in one or more locations within a host cell.

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

[0075] The term "metastasis" as used herein refers to all CTLA4-involved processes that support cancer cells to disperse from a primary tumor, infiltrate lymphatic and / or blood vessels, circulate through the bloodstream, and grow at distant foci (metastases) in normal tissues elsewhere in the body. In particular, it refers to cellular events of tumor cells, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, stimulated or mediated by non-catalytic or catalytic activity of CTLA4 (preferably including CTLA4 phosphorylation and / or CTLA4-mediated signaling) and underlying metastasis.

[0076] The term "microenvironment" as used herein means any part or region of a tissue or body that has permanent or temporary, physical or chemical differences from other regions of the tissue or body. In the case of tumors, the term "tumor microenvironment" as used herein refers to the environment in which the tumor resides, the acellular regions within the tumor, and the regions immediately outside the tumor tissue but not related to the intracellular compartments of the cancer cells themselves. Tumors and tumor microenvironment are closely related and constantly interact. Tumors can change their microenvironment, and the microenvironment can affect tumor growth and spread. Typically, tumor microenvironments have a low pH, within the range of 5.0-7.0, or within the range of 5.0-6.8, or within the range of 5.8-6.8, or within the range of 6.2-6.8. On the other hand, normal physiological pH is in the range of 7.2-7.8. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients, but higher concentrations of lactate, compared to plasma. In addition, the tumor microenvironment can have a temperature that is 0.3-1°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 incorporated by reference in its entirety. The term "non-tumor microenvironment" refers to the microenvironment at a site other than a tumor.

[0077] The term "monoclonal antibody" as used herein 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, but excludes variant antibodies that may, for example, contain naturally occurring mutations or arise during the production of a monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies specific for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is specific for a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies and should not be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

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

[0079] The term "natural antibody" as used herein refers to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (V), also called the variable heavy domain or heavy chain variable domain. 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 (V), also called the variable light domain or light chain variable domain.L ), followed by the constant light chain (C L The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

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

[0081] The term "percent (%) amino acid sequence identity" as used herein 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 in a reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using ALIGN-2, a sequence comparison computer program. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office (Washington DC, 20559), 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 must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0082] In situations where ALIGN-2 is used for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity with or to 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 by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used herein are obtained as described in the previous section using the ALIGN-2 computer program.

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

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

[0085] The terms "purified" and "isolated" as used herein refer to an antibody or nucleotide sequence according to the 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 of the same type of biopolymer is present, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode a polypeptide, although the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0086] The term "recombinant antibody" as used herein refers to an antibody (e.g., a chimeric, humanized or 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 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.

[0087] The term "CTLA4" as used herein refers to an immune checkpoint having an amino acid sequence described in U.S. Patent Nos. 5,434,131, 5,844,095, and 5,851,795, or any part or derivative thereof, which recognizes and binds to or interferes with B7, blocking its binding to CD28 and / or CTLA4 (e.g., endogenous CD28 and / or CTLA4). In certain embodiments, the extracellular domain of wild-type CTLA4 starts with a methionine at position +1 and ends with an aspartic acid at position +124, or the extracellular domain of wild-type CTLA4 starts with an alanine at position -1 and ends with an aspartic acid at position +124. Wild-type CTLA4 is a cell surface protein with an N-terminal extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic domain. The extracellular domain binds to a target molecule, for example, a B7 molecule. In cells, naturally occurring wild-type CTLA4 protein is translated as an immature polypeptide containing a signal peptide at the N-terminus. The immature polypeptide undergoes post-translational processing, including cleavage and removal of the signal peptide, to generate a CTLA4 cleavage product with a newly generated N-terminus that differs from the N-terminus of the immature form. Those skilled 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, generating a molecule that starts before the common starting amino acid methionine. Thus, mature CTLA4 protein may start with methionine at position +1 or alanine at position -1. The mature form of the CTLA4 molecule includes the extracellular domain or any part thereof.

[0088] The term "therapeutically effective amount" of an antibody of the present invention means an amount of the antibody sufficient to treat the cancer in question, at 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 dose 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 used, the specific composition used, the age, weight, general health, sex, and diet of the patient, the time of administration, route of administration, and excretion rate of the specific antibody used, the duration of treatment, drugs used in combination or concomitantly with the specific antibody used, and similar factors known in the medical art. For example, it is known in the art to begin administering a compound at a level lower than that required to achieve the desired therapeutic effect, and 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 A heterodimer, which usually consists of a gene encoding a V H and V L "dsFv" refers to a gene fusion containing a V stabilized by a disulfide bond. H ::V L Heterodimers. Divalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by linking monovalent scFvs with peptide linkers (such as bivalent sc(Fv)2).

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

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

[0092] The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) have a generally similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H or V L The V domain may be sufficient to confer specificity of antigen binding. H or V L The V domains are used to isolate antibodies that bind to a particular antigen, each of which is associated with a complementary V L or V HLibraries of domains can be screened. See, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.

[0093] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively 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 exemplary embodiments. Although certain embodiments of the present invention are specifically described herein, those skilled in the art will readily appreciate that the same principles are equally applicable to and may be used in other systems and methods. Before describing the disclosed embodiments of the present invention in detail, it should 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 purposes of description and not limitation. Furthermore, although certain methods are described with reference to steps presented herein in a particular order, in many cases these steps can be performed in any order as would be understood by one skilled in the art, and thus the novel methods are 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. Additionally, 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 properties such as amounts of ingredients, molecular weights, percentages, ratios, reaction conditions, and the like used in the specification and claims should be understood to be 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 the specification and claims are approximations that may vary depending on 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 scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present 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 understood that each component, compound, substituent, or parameter disclosed herein is to be construed as disclosed for use alone or in combination with one or more of each other component, compound, substituent, or parameter disclosed herein.

[0098] Also, each amount / value or amount / value range of each component, compound, substituent, or parameter disclosed herein should be construed as being disclosed in combination with each amount / value or amount / value range disclosed for any other component(s), compound(s), substituent, or parameter(s) disclosed herein, and thus, for purposes of this description, any combination of amounts / values ​​or amount / value ranges for two or more component(s), compound(s), substituent(s), or parameters disclosed herein should also be understood to be disclosed in combination with each other.

[0099] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Thus, a disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges should be interpreted as a 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 examples should be interpreted as a disclosure of either a lower or upper limit of a range, and thus can be combined with any other lower or upper limit or specific amount / value of a range of the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range of that component, compound, substituent, or parameter.

[0100] A. Anti-CTLA4 antibody In one aspect, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to a human CTLA4 protein. The isolated heavy chain variable region polypeptide comprises three complementarity determining regions, 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), In the formula, 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] FIG. 1 shows an alignment of exemplary isolated heavy chain variable regions of the invention, with the complementarity determining regions H1, H2, and H3 boxed.

[0102] In another aspect, the present 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, L1 arrangement 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), In the formula, X9 is A or I, and X 10 is Y, S or H, and X 11 is V or G, and X 12 is V or I.

[0103] FIG. 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 polypeptides and isolated light chain variable region polypeptides of the present invention were each derived from a parent antibody using the methods disclosed in U.S. Patent No. 8,709,755. Methods for producing the isolated heavy chain variable region polypeptides and isolated light chain variable region polypeptides, as well as methods for producing antibodies and antibody fragments, are disclosed in U.S. Patent No. 8,709,755 and are hereby incorporated by reference herein.

[0105] In another embodiment, the invention comprises the heavy chain variable region shown in Figure 1 and the light chain variable region shown in Figure 2. The amino acid sequences of the 16 heavy chain variable regions of Figure 1 are set forth 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 of Figure 1 are set forth 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 and light chain variable regions can specifically bind to human CTLA4. Antibodies or antibody fragments that contain 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 a non-tumor microenvironment (e.g., pH 7.4). As a result, 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 a normal tissue microenvironment.

[0106] Thus, the anti-CTLA4 antibodies or antibody fragments of the present invention have reduced side effects as well as comparable efficacy compared to monoclonal anti-CTLA4 antibodies known in the art due to their reduced binding affinity to CTLA4 in normal tissue microenvironments. These characteristics allow higher doses of these anti-CTLA4 antibodies or antibody fragments to be delivered to patients, thus providing a more effective treatment option.

[0107] The present invention includes heavy and light chain variable regions shown in Figures 1-2, as well as those having the amino acid sequences of SEQ ID NOs: 7-38, but 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 and light chain variable regions outside the complementarity determining regions can be mutated according to the principles of substitution, insertion and deletion discussed in this application. In still further embodiments, the constant regions can be modified to provide variants.

[0108] In deriving these variants, the process described herein serves as a guide. The variants of the heavy and light chain variable regions can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy and light chain variable regions or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequences of the heavy and light chain variable regions. Any combination of deletions, insertions, and substitutions can be made to arrive at the antibody or antibody fragment 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 are provided that have one or more amino acid substitutions. Sites of interest for substitution mutagenesis include the CDRs and framework regions (FRs). Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions". More substantial changes are provided in Table 1 under the heading of "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 products may be screened for a desired activity, e.g., retention / improvement of antigen binding, or reduced immunogenicity. [Table 1]

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

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

[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 substantially retained. An exemplary substitution variant is an affinity matured antibody, which may be conveniently generated using techniques of phage display-based affinity maturation, such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0113] For example, modifications (e.g., substitutions) may be made in the CDRs to improve antibody affinity. Such modifications can be made in CDR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in the SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting from a secondary library 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 genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-specific approach in which several 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 may 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 to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may be outside the "hot spots" of the CDRs or SDRs. Variants V as provided above may be modified in any manner that would reduce the binding affinity of the antibody or antibody fragment. H and V L In certain embodiments of the sequences, each CDR is either unaltered or does not contain one, two 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, a target residue or group 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 of the antibody or antibody fragment with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex identifies contact points between the antibody or antibody fragment and the antigen. Such contact and adjacent residues may be targeted or eliminated as candidates for substitution. The variants may be screened to determine whether they contain the desired properties.

[0116] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions (ranging in length from one residue to polypeptides of 100 or more residues), as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibodies include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0117] Amino acid sequence modification(s) 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. H and V L Only the CDRs in the V of a human antibody are H and V LIt is known that when a humanized antibody is produced by grafting FRs in the V region of a non-human antibody, the antigen-binding activity is reduced compared to that of the original antibody derived from a non-human animal. H and V L Some amino acid residues in the V region of a human antibody are thought to be directly or indirectly related to the antigen-binding activity. H and V L Substitution of different amino acid residues from the FR of the human antibody would result in decreased binding activity. To solve this problem, the V of the human antibody in the antibody grafted with human CDRs should be H and V L In the amino acid sequence of the FR of the antibody, it is necessary to attempt to identify amino acid residues that are directly involved in binding to the antibody, or that interact with amino acid residues of the CDR, or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. The reduced antigen-binding activity can be increased by substituting the identified amino acids with amino acid residues of the original antibody derived from a non-human animal.

[0118] Modifications and changes can be made in the structure of the antibodies of the invention and the DNA sequences which encode them and still obtain a functional molecule which encodes an antibody having the desired characteristics.

[0119] When making changes to amino acid sequences, the hydrophilicity index of amino acids may be taken into consideration. The importance of the hydrophilicity amino acid index in conferring interactive biological functions to proteins is generally understood in the art. It is accepted that the relative hydrophilicity characteristics of amino acids 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 hydrophobicity and charge characteristics: 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] Further objects of the present invention also include function-conservative variants of the antibodies of the invention.

[0121] A "function-conservative variant" is one in which a given amino acid residue in a protein or enzyme has been altered without altering the overall conformation and function of the polypeptide, including, but not limited to, amino acid substitutions with amino acids having similar properties (e.g., polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those designated as conserved amino acids may differ in a protein, resulting in a change in the percent sequence similarity of the protein or amino acids between any two proteins with similar functions, which may be, for example, 70%-99% when the similarity is determined according to an alignment scheme such as a cluster method based on the MEGALIGN algorithm. A "function-conservative variant" also includes a polypeptide having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95%, as determined by BLAST or FASTA algorithms, and has 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 more than about 90%, preferably more than 95%, are similar (functionally identical) over the entire length of the shorter sequence. Preferably, 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, etc.

[0123] For example, certain amino acids can be substituted by other amino acids in a protein structure without significant loss of activity. Because the ability and nature of protein interaction defines the biological functional activity of a protein, certain amino acid substitutions can be made in a protein sequence, and of course in its DNA coding sequence, to nevertheless obtain a protein with similar properties. Thus, it is contemplated that various changes can be made in the sequence of the antibody or antibody fragment of the present invention, or in the corresponding DNA sequence encoding said antibody or antibody fragment, without significant loss of their biological activity.

[0124] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydrophilicity index or score and still result in a protein having a similar biological activity, i.e., still resulting in a biologically functional equivalent protein.

[0125] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various characteristics discussed above are known to those of skill 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 extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0127] If the antibody comprises an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, generally linked by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharides can comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody of the present invention can be made to generate antibody variants with specific improved properties.

[0128] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues), although Asn297 may be located about ±3 amino acids upstream or downstream of position 297, i.e., at positions 294-300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.) and 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 / 01321, and US2004 / 01321. 40, 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, and Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986; U.S. Patent Publication No. US2003 / 0157108A; and WO2004 / 056312A1 (especially Example 11)), as well as knockout cell lines, such as alpha-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] Further provided are antibody variants comprising biantennary oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are 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, U.S. Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached 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 variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0131] In certain embodiments, the present invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important, but where certain effector functions (such as ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression 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 to assess ADCC activity of a molecule of interest include those described in U.S. Pat. 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. Pat. 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.). Useful effector cells for 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, for example in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays may be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods, vol.202, pp.163-171, 1996; Cragg, MS et al., Blood, vol.101, pp.1045-1052, 2003; and Cragg, MS, and MJGlennie, Blood, vol.103, pp.2738-2743, 2004). Also, FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l.Immunol., vol.18, pp.1759-1769, 2006).

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

[0133] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 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 with 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, modifications are made in the Fc region to result in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC). For example, as described in 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), involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol., vol.117, pp.587-593, 1976 and Kim et al., J.Immunol., vol.24, p.249, 1994) and increased half-life are described in US2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the following residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution at residue 434 in 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 WO 94 / 29351.

[0137] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replacement of residues occurs at sites accessible to the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, and the antibody can be conjugated to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. 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 ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0138] antibody derivative In certain embodiments, the antibody or antibody fragment provided herein may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies or antibody fragments 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, 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-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, 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 may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody or antibody fragment may vary, and when two or more polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may 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 will be used in therapy under a given condition, etc.

[0139] In another embodiment, a conjugate of an antibody or antibody fragment and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. 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, including but not limited to wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells in close proximity to the antibody-non-proteinaceous moiety.

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

[0142] In certain embodiments, the anti-CTLA4 antibodies or antibody fragments of the invention have a potency under conditions in the tumor microenvironment 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~10 -13 M, or 10 -9 M~10 -13 In one embodiment, the antibody or antibody fragment has a dissociation constant (Kd) with CTLA4 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.

[0143] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed on the antibody of interest and a Fab version of its antigen using the following assay: The solution binding affinity of a Fab for an antigen is determined by binding the Fab to a minimal concentration of ( 125 I) Equilibration with labeled antigen and then capture of bound antigen using plates coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I]-antigen is mixed 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)). The Fab of interest is then incubated overnight, although incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). A concentration of each Fab that gives 20% or less of maximum binding is selected for use in competitive binding assays.

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

[0145] The anti-CTLA4 antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, an anti-CTLA4 antibody fragment is used, such as an Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragment formed from an antibody fragment, and a multispecific antibody. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody, or other antibody class or isotype as 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, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), also WO 93 / 16185, and U.S. Patent 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 increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0146] The diabody of the present invention can be bivalent or bispecific.For example, 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 tetrabodies 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, comprising all or a portion of the heavy chain variable domain, or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1).

[0148] Antibody fragments can be produced 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 phages) as described herein.

[0149] In some embodiments, the anti-CTLA4 antibody of the present invention may 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, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a 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. The chimeric antibody includes an antigen-binding fragment thereof.

[0150] In certain embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains, in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to 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. 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"), as well as Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0152] Human framework regions that may 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 a particular subgroup 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 (somatically 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. Immunol., vol. 151, p. 2623, 1993). 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 antibody of the present invention is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity 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, a bispecific antibody can bind to two different epitopes of CTLA4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CTLA4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0154] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983; WO 93 / 08829; and Traunecker et al., EMBO J. vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), and / or using glycerol-binding proteins to generate glycerol-binding fragments (see, e.g., Hollinger et al., J. Immunol., vol. 148, pp. 1547-1553, 1992). al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993), and by 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 (see, for example, Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991).

[0155] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, eg, US 2006 / 0025576 A1).

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

[0157] The physical / chemical properties and / or biological activity 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 which are described in U.S. Patent No. 8,853,369.

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

[0159] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), in which an antibody or antibody fragment is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0425235 B1), auristatins such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (U.S. Pat. No. 5,635,483, Nos. 5,780,588 and 7,498,298), dolastatins, calicheamicin or derivatives thereof (see U.S. Pat. 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. 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., al.,Bioorg.& Med.Chem.Letters,vol.12,vol.1529-1532,2002, King et al.,J.Med.Chem.,vol.45,pp.4336-4343,2002, 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, nonbinding 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 proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

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

[0162] Conjugates of antibodies / antibody fragments and cytotoxic agents can be made using a variety of 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-azido 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 radionucleotides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992, U.S. Patent No. 5,208,020) can be used.

[0163] Immunoconjugates herein include, but are not limited to, 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), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA), but explicitly contemplate conjugates prepared with cross-linking reagents.

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

[0165] An exemplary ADC is Ab-(LD) p where p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to an 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 with 1, 2, 3, or 4 engineered 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 modification, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to a 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 A "linker" (L) is bifunctional or multifunctional that 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, an ADC can be prepared using a linker that has reactive functionality for covalently binding to a drug and an antibody. For example, in some embodiments, a cysteine ​​thiol of an antibody or antibody fragment (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to generate an ADC.

[0167] In one embodiment, the linker has a functionality that can react with the free cysteine ​​present on the antibody to form a covalent bond.Non-limiting examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated ester such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate.See, for example, the conjugation method in Klussman, et al, Bioconjugate Chemistry, vol.15, pp.765-773, 2004, p.766.

[0168] In some embodiments, the linker has a functionality that can react with an electrophilic group present on an 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 an antibody to form a covalent bond to an antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0169] The linker may comprise one or more linker moieties. Exemplary linker moieties 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 moieties are known in the art, some of which are described below.

[0170] The linker can be a "cleavable linker" that facilitates the release of the drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (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 ADCs having the following formula II as linkers: Ab-(A), where A is an "extender unit", a is an integer from 0 to 1, W is an "amino acid unit", w is an integer from 0 to 12, and Y is a "spacer unit", where y is 0, 1, or 2. ADCs containing linkers of formula II have the formula I(A): Ab-(A a -W w -Y y -D) p where Ab, D, and p are defined above for formula I. Exemplary embodiments of such linkers are described in U.S. Patent No. 7,498,298.

[0172] In some embodiments, a linker component comprises an "extender unit" (A), which links the antibody to another linker component or to a drug moiety. Non-limiting exemplary extender units are shown below (the wavy line indicates the site of covalent attachment to an antibody, drug, or additional linker component): [ka]

[0173] In some embodiments, the linker component comprises an "amino acid unit" (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to an intracellular protease (e.g., a lysosomal enzyme) (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 units can include naturally occurring amino acid residues and / or minor amino acids and / or non-natural amino acid analogs, such as a citrulline amino acid unit 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-type linker can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such a peptide bond can be prepared, for example, according to a liquid phase synthesis method (e.g., E. Schroder and K. Lubke (1965) "The Peptides", volume 1, pp 76-136, Academic Press).

[0175] In some embodiments, the linker component comprises a "spacer unit" (Y), which links the antibody to the drug moiety either directly or via an extender unit and / or an amino acid unit. The spacer unit may 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, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by tumor cell-associated proteases 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, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0176] A "self-immolative" spacer unit allows for 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 attached to the amino acid unit via an amide bond, generating a carbamate, methyl carbamate, 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-immolative linker has the following structure: [ka] wherein Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano, m is an integer ranging from 0 to 4, X can be one or more additional spacer units or can be 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 exemplary X spacer units are: [ka] 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-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. 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, spacers that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol.2, pp.223-, 1995), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J.Amer.Chem.Soc., vol.94, p.5815-, 1972), and 2-aminophenylpropionic acid amides (Amsberry et al., J.Org.Chem., vol.55, p.5867, 1990). Attachment of a drug to the alpha carbon of a glycine residue is another example of a self-immolative spacer that can be useful in ADCs (Kingsbury et al., J.Med.Chem., vol.27, p.1447, 1984).

[0178] In some embodiments, the linker L may be a dendritic linker for covalently linking 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). The dendritic linker can increase the molar ratio of drug to antibody (i.e., the loading associated with the potency of the ADC). Thus, when an antibody has only one reactive cysteine ​​thiol group, multiple drug moieties can be attached via the dendritic linker.

[0179] In the context of ADCs, a non-limiting exemplary linker is shown in Formula I below: [ka] wherein R1 and R2 are independently selected from H and C1-C6 alkyl. In some embodiments, R1 and R2 are each -CH3. [ka] In the formula, n is 0 to 12. In some embodiments, n is 2 to 10. In some embodiments, n is 4 to 8.

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

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

[0182] The compounds of the invention are expressly contemplated to be used to prepare ADCs using, but not limited to, the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy)succinimide ester (EMCS), N-[γ-maleimidobutyloxy]succinimide ester (GMBS), 1,6-hexane-vinylsulfone (HBVS), succinimide, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succindiyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2-aminophenyl)propionate (SPA), 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), succindiyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2-aminophenyl)propionate (SPA), succinimidyl 4 ... 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: dithiobismaleimidoethane (DTME), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (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-azido 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, bis-maleimide reagents allow for the attachment of thiol groups of cysteines of antibodies to thiol-containing drug moieties, linkers, or linker-drug intermediates. Other functional groups that react with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates;

[0183] Certain useful linker reagents can be obtained from a variety of 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-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See, e.g., WO 94 / 11026.

[0185] ii) Exemplary Drug Moieties 1) Maytansine and maytansinoids In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, which are mitotic inhibitors that act by inhibiting the polymerization of tubulin. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinoids are described, for example, in U.S. Pat. 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, and 4,309,429. Nos. 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] Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because: (i) they are relatively accessible to preparation by fermentation or chemical modification or derivatization of fermentation products; (ii) they are amenable to derivatization with functional groups suitable for conjugation via non-sulfide linkers to antibodies; (iii) they are stable in plasma; and (iv) they are effective against a variety of tumor cell lines.

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

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

[0189] Exemplary maytansinoid drug moieties also include those having modifications such as C-9-SH (U.S. Pat. No. 4,424,219) (prepared, for example, by reaction of maytansinol with H2S or P2S5), C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (prepared, for example, from Nocardia), C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by conversion of maytansinol by Streptomyces), C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared, for example, from Trewia nudlflora), C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, for example, by demethylation of maytansinol with Streptomyces sp.), and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by titanium trichloride / LAH reduction of maytansinol).

[0190] Many positions on a maytansinoid compound are useful as attachment positions. 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 with a hydroxyl group, the C-14 position with a hydroxymethyl modification, the C-15 position with a hydroxyl group, and the C-20 position with a hydroxyl group. In some embodiments, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0191] Maytansinoid drug moieties include those having the following structure: [ka] where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the ADC. Each R can independently be H or a C1-C6 alkyl. The alkylene chain linking the amide group to the sulfur atom can be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Pat. Nos. 633,410 and 5,208,020; Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; Liu et al., Proc. Nall. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996).

[0192] All stereoisomers of the maytansinoid drug moiety are contemplated for the ADCs of the invention, i.e., any combination of R and S configurations at the chiral carbon (U.S. Pat. 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: [ka]

[0193] Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, DM1, DM3, and DM4, which have the following structures: [ka] Here, the wavy line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate.

[0194] An exemplary antibody-drug conjugate in which DM1 is linked to a thiol group of an antibody via a BMPEO linker has the following structure and abbreviations: [ka] where Ab is an antibody, n is 0, 1, or 2, and p is 1 to about 20. In some embodiments, p is 1 to 10, or 1 to 7, or 1 to 5, or 1 to 4.

[0195] Immunoconjugates comprising 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, US 2005 / 0276812 A1, and EP 0425235 B1. 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, antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020. In some embodiments, ADCs conjugated with an average of 3-4 maytansinoid molecules per antibody molecule have shown efficacy in enhancing cytotoxicity of target cells without adversely affecting antibody function or solubility. In some cases, even one molecule of toxin / antibody is expected to enhance cytotoxicity over the use of naked antibodies.

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

[0198] (2) Auristatins and dolastatins Drug moieties include dolastatins, auristatins, and analogs and derivatives thereof (U.S. Patent Nos. 5,635,483, 5,780,588, 5,767,237, and 6,124,431). Auristatins are derivatives of dolastatin-10, a marine mollusc compound. Without intending to be bound by any particular theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001), as well as anticancer (U.S. Pat. No. 5,663,149), and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The dolastatin / auristatin drug moiety may be attached to the antibody through the N-terminus or the C-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 N-terminally linked monomethyl auristatin drug moieties, D, as disclosed in U.S. Pat. Nos. 7,498,298 and 7,659,241. E and D. F Examples include: [ka] In the formula, D E and D. F The wavy lines indicate the sites of covalent attachment to the antibody or antibody-linker moiety, and independently at each position: R 2 is selected from H and C1-C8 alkyl; R 3is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle); R 4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle); R 5 is selected from H and methyl; Or, R 4 and R 5 Together they form a carbocyclic ring, and the formula is -(CR a R b ) n -R a and R b is independently selected from H, C1-C8 alkyl, and C3-C8 carbocycle; n is selected from 2, 3, 4, 5, and 6; R 6 is selected from H and C1-C8 alkyl; R 7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle); Each R 8 is independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle, and O-(C1-C8 alkyl); R 9 is selected from H and C1-C8 alkyl; R 10 is selected from aryl or a C3-C8 heterocycle; Z is O, S, NH, or NR 12 and R 12 is C1-C8 alkyl, R 11 H, C1~C 20 Alkyl, aryl, C3-C8 heterocycle, -(R 13 O)m ~R1 4 , or -(R 13 O) m ~CH(R 15 )2 are selected, m is an integer ranging from 1 to 1000; R 13 is a C2-C8 alkyl; R 14 is H or C1-C8 alkyl, e is independently at each occurrence H, COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, or -(CH2) n -SO3-C1-C8 alkyl; e, independently at each occurrence, is H, C1-C8 alkyl, or -(CH2) n -COOH, R 18 is -C(R 8 )2-C(R 8 )2-aryl, -C(R 8 )2-C(R 8 )2-(C3-C8 heterocycle), and -C(R 8 )2-C(R 8 )2-(C3-C8 carbocyclic ring); n is an integer ranging from 0 to 6.

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

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

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

[0203] In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 2 and R 6 are methyl, and 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 Ha-(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 auristatin embodiment of is MMAE, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0211] Formula D E An exemplary auristatin embodiment of is MMAF, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0212] Other exemplary embodiments include monomethylvaline compounds having a carboxy modification of the phenylalanine at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethylvaline compounds having a side chain modification of the phenylalanine at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0213] Non-limiting exemplary embodiments of the ADC of formula I that comprises MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.It has been shown that immunoconjugates that comprise MMAF linked to antibody by a non-proteolytic cleavable linker have comparable activity to immunoconjugates that comprise MMAF linked to antibody by a proteolytic cleavable linker (Doronina et al., Bioconjugate Chem., vol.17, pp.114-124, 2006).In some such embodiments, it is believed that drug release is affected by the degradation of antibody in cells.

[0214] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (see, for example, E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties, in some embodiments, can be prepared according to the methods of U.S. Pat. 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 compound of formula D, such as MMAE, E auristatin / dolastatin drug moieties, and D such as MMAF E Drug-linker intermediates and derivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, as well as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAE, can be prepared using methods described in U.S. Pat. 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 then conjugated to an 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 its analogs can generate double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; Lode et al., Cancer Research, vol. 58, pp. 2925-2928, 1998). Although calicheamicin has an intracellular site of action, in certain cases it does not readily cross the cell membrane. Thus, in some embodiments, cellular uptake of these agents through antibody-mediated internalization can greatly enhance their cytotoxic effects. Non-limiting exemplary methods for preparing antibody-drug conjugates having a calicheamicin drug moiety are described, for example, in U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, and 5,767,285.

[0217] (4) Pyrrolobenzodiazepines In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin, PBD, 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). Several PBDs, both natural and analogues, have since been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994), including dimers of a tricyclic PBD backbone (U.S. Pat. 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, it is believed that the dimeric structure confers the proper three-dimensional shape for isohelicity with the minor groove of B-form DNA, fitting snugly 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 with 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] PBD dimers have been conjugated to antibodies and the resulting ADCs have been shown to have anti-cancer properties. Non-limiting exemplary linking sites on the PBD dimer include the 5-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (WO2009 / 016516, US2009 / 304710, US2010 / 047257, US2009 / 036431, US2011 / 0256157, WO2011 / 130598).

[0219] Non-limiting exemplary PBD dimer components of ADCs include: [ka] or a salt or solvate thereof, wherein The wavy line indicates the site of covalent attachment to the linker; The dotted lines indicate the presence of an optional double bond between C1 and C2 or between C2 and C3; R 2 are independently H, OH, =O, =CH2, CN, R, OR, =CH-R D , =C(R D )2, O—SO2—R, CO2R, and COR, optionally further selected from halo or dihalo; R D is independently selected from R, COR, COR, CHO, COH, and halo; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn, and halo; R 7 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn, and halo; Q is independently selected from O, S, and NH; R 11 is either H, or R, or, if Q is O, SO3M, where M is a metal cation; R and R′ are each independently an optionally substituted C 1~8 Alkyl, C1~12 Alkyl, C 3~8 Heterocyclyl, C 3~20 Heterocycles, and C 5~20 aryl groups, optionally together with the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring; R 12 , R 16 , R 19 and R 17 are R 2 , R 6 , R 9 and R 7 as defined for R'' is C3~ 12 an alkylene group, the chain of which may be interrupted by one or more heteroatoms, such as O, S, N(H), NMe, and / or aromatic rings (e.g., benzene or pyridine), which rings are optionally substituted; X and X' are independently selected from O, S and N(H).

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

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

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

[0223] In some embodiments, R 2 and / or R 12 =CH-R D When it is, each group can independently have any of the configurations shown below: [ka] In some embodiments, a=CH-R D is in structure (I). In some embodiments, R″ is a C3 alkylene group or a C5 alkylene group.

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

[0225] PBD dimers and ADCs containing PBD dimers can be prepared according to methods known in the art, see, e.g., WO2009 / 016516, US2009 / 304710, US2010 / 047257, US2009 / 036431, US2011 / 0256157, WO2011 / 130598.

[0226] (5) Anthracycline In some embodiments, the ADC may include an anthracycline. An anthracycline is an antibiotic compound that exhibits cytotoxic activity. Without intending to be bound by any particular theory, studies have shown that anthracyclines may act to kill cells by several different mechanisms, including: 1) intercalation of the drug molecule into the cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) drug-induced production of free radicals that then react with cellular macromolecules to cause damage to the cell; and / or 3) interaction of the drug molecule with cell membranes (see, for example, C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; NR Bachur, "Free Radical Damage" (ibid., pp. 97-102)). These potentially cytotoxic anthracyclines have been used to treat a number of cancers, including leukemia, breast cancer, lung cancer, ovarian adenocarcinoma and sarcoma (see, for example, PH-Wiernik, in Anthracycline: Current Status And New Developments, p. 11).

[0227] Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (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., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, ...). (see, for example, Saleh 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 reacts specifically 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 semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycosidic amino of doxorubicin and is undergoing clinical evaluation (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 / phase III trials for hepatocellular carcinoma (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Abs 1448, 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 antibodies via a variety of linkers, including several linkage sites and the linkers described herein (US2011 / 0076287, WO2009 / 099741, US2010 / 0034837, WO2010 / 009124).

[0230] The linker of PNU-159682-maleimide acetal-Ab is acid labile, whereas PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer (R 1 R 2 The linker in the )-Ab is protease cleavable.

[0231] (6) Other drug moieties Drug moieties also include 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 enzymatically active toxins and fragments thereof, including but not limited to diphtheria A chain, nonbinding active fragments 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, Phytolacca Also included are the americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichotheenes. See, e.g., WO93 / 21232.

[0232] Drug moieties also include compounds that have nucleolytic activity (eg, ribonucleases or DNA endonucleases).

[0233] In certain embodiments, the immunoconjugate may include a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212In some embodiments, when the immunoconjugate is used for detection, it is preferably used with a radioactive atom for scintigraphy studies, e.g., Tc 99 Or I 123 or spin labels 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 may be complexed to various metal chelators, and conjugated to antibodies, for example, for PET imaging (WO2011 / 056983).

[0234] Radiolabels or other labels may be incorporated into the immunoconjugates by known methods. For example, peptides may be biosynthesized or chemically synthesized using suitable amino acid precursors that contain, for example, one or more fluorine-19 atoms in place of one or more hydrogens. In some embodiments, Tc 99 , I 123 , Re 186 , Re 188 , and In 111 Labels such as , can be attached via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be attached via lysine residues of 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 (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) to an active drug, such as an anti-cancer drug. Such immunoconjugates are, in some embodiments, useful in antibody-dependent enzyme-mediated prodrug therapy ("Adept"). Enzymes that may be conjugated to an antibody include, but are not limited to, alkaline phosphatase, useful for converting phosphate-containing prodrugs to free drugs, arylsulfatases, 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 useful for converting peptide-containing prodrugs to free drugs, such as Serratia protease, pyrolysis, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), D-aminopeptidases, and the like. Examples of enzymes include D-alanyl carboxypeptidase, useful for converting prodrugs containing acid substituents, carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, useful for converting glycosylated prodrugs to free drugs, β-lactamase, useful for converting drugs derivatized with β-lactams to free drugs, and penicillin amidases, such as penicillin V amidase and penicillin G amidase, useful for converting drugs derivatized with amine nitrogens bearing phenoxyacetyl or phenylacetyl groups to free drugs. In some embodiments, the enzymes can be covalently bound 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 Load Drug loading is represented by the average number p of drug moieties per antibody in the molecule of formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. The ADC of formula I includes a collection of antibodies conjugated with drug moieties ranging from 1 to 20. The average number of drug moieties per antibody used to prepare 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 that is different from ADCs with other drug loads, separation, purification, and characterization of the homogeneous ADC can be achieved by means such as reverse phase HPLC or electrophoresis.

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

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

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

[0240] It should be understood that when two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADCs with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay that is specific for the antibody and specific for the drug. 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, homogeneous ADCs with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0241] iv) Particular methods for preparing immunoconjugates Immunoconjugates that are ADCs of formula I can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: (1) reacting a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety, D, and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent to form DL via a covalent bond, followed by reaction with a nucleophilic group of an antibody. An exemplary method for preparing ADCs of formula I via the latter route is described in U.S. Pat. No. 7,498,298.

[0242] Nucleophilic groups on an antibody include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups on which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) active esters, such as NHS esters, HOBt esters, haloformates, 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. Antibodies can be made reactive for conjugation with linker reagents by treating with a reducing agent, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), 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 antibodies through modification of lysine residues, for example by reacting them with 2-iminothiolane (Traut's reagent) to convert amines to thiols. Reactive thiol groups can also be introduced into antibodies 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 produced by reaction between electrophilic groups on an antibody or antibody fragment, such as aldehyde or ketone carbonyl groups, and nucleophilic groups on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugar of a 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 on a 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, reaction of carbohydrate moieties of glycosylated antibodies with either galactose oxidase or sodium metaperiodate can result in carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing N-terminal serine or threonine residues can be reacted 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 drug moieties or linker nucleophiles.

[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 covalent bonds with electrophilic groups 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 and benzyl halides, such as haloacetamides, (iii) aldehydes, ketones, carboxyls, and maleimide groups.

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

[0246] In yet another embodiment, an antibody or antibody fragment may be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, and the antibody / antibody fragment-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

[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 breast, pancreatic, esophageal, lung and / or brain cells or tissues.

[0248] A further aspect of the present invention relates to an anti-CTLA4 antibody or antibody fragment of the present invention for diagnosing and / or monitoring cancer or another disease in which CTLA4 expression levels are increased or decreased from normal physiological levels at at least one location in the body.

[0249] In a preferred embodiment, the antibody or antibody fragment of the present 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 antibody or antibody fragment of the present 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, may also 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 in the patient is detected. Any suitable known method may 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 can be useful for the diagnosis and stage of cancer and disease associated with overexpression of CTLA4.The cancer associated with overexpression of CTLA4 can include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulva cancer, thyroid cancer, hepatocellular carcinoma, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer, or other CTLA4 expressing or overexpressing hyperproliferative disease.

[0251] The antibody or antibody fragment of the present invention may be useful for the diagnosis of diseases other than cancer in which CTLA4 expression is increased or decreased. Both soluble and cellular forms of CTLA4 can be used for such diagnosis. Typically, such diagnostic methods involve the use of biological samples obtained from patients. Biological samples include various sample types obtained from subjects that can be used for diagnostic or monitoring assays. 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, biological samples include cells obtained from tissue samples collected from individuals suspected of cancer associated with CTLA4 overexpression, and in preferred embodiments, cells obtained from glioma, 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 a particular embodiment, the invention is a method for diagnosing cancer associated with CTLA4 overexpression in a subject by detecting CTLA4 on cells from the subject using an antibody of the invention. In particular, the method comprises the following steps: (a) contacting a biological sample of a subject with an antibody or antibody fragment according to the invention under conditions suitable for the antibody or antibody fragment to form a complex with cells in the biological sample that express CTLA4; (b) detecting and / or quantifying the complex, wherein detection of the complex is indicative of a cancer associated with overexpression of CTLA4.

[0253] To monitor the progression of cancer, the methods according to the invention may be repeated at different times to determine whether antibody binding to the sample increases or decreases, from which it can be determined whether the cancer has progressed, regressed or stabilized.

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

[0255] In one embodiment, an anti-CTLA4 antibody or antibody fragment is provided for use in a method of diagnosis or detection. In a further aspect, a method is provided for detecting the presence of CTLA4 in a biological sample. In a further aspect, a method is provided for quantifying the amount of CTLA4 in a biological sample. In a particular embodiment, the method comprises contacting a biological sample with an anti-CTLA4 antibody or antibody fragment described herein under conditions that allow 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 a method 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, the treatment comprises administering an anti-CTLA4 antibody or antibody fragment to a subject.

[0256] In certain embodiments, labeled anti-CTLA4 antibodies or antibody fragments are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected (e.g., by 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 (such as rare earth chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.), luciferases (such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase, xanthine oxidase, etc.), coupled enzymes that oxidize dye precursors using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, bacteriphage labels, stable free radicals, etc.

[0257] D. Pharmaceutical Preparations Anti-CTLA4 antibodies or antibody fragments have cell-killing activity. This cell-killing activity extends to multiple different types of cell lines. In addition, these antibodies or antibody fragments, once conjugated to cytotoxic agents, can reduce tumor size and exhibit reduced toxicity. Therefore, anti-CTLA4 antibodies, their fragments or immunoconjugates can be useful for treating proliferative diseases associated with the expression of CTLA4. The antibodies, fragments or immunoconjugates can be used alone or in combination with any suitable drug or other conventional treatment.

[0258] Anti-CTLA4 antibody or antibody fragment can be used to treat diseases related to expression, overexpression or activation of CTLA4.There is no particular limit to the type of cancer or tissue that can be treated without the requirement of CTLA4 expression.Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, 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 glioma, 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 innate immune responses and can therefore be used to treat human immune diseases such as sepsis. Anti-CTLA4 antibodies or antibody fragments of the present invention can also be used as adjuvants for immunization, such as vaccines, and as anti-infective agents against, for example, bacteria, viruses, and parasites.

[0260] Anti-CTLA4 antibodies or antibody fragments can be used to protect against, prevent or treat thrombotic diseases such as venous and arterial thrombosis and atherothrombosis. Anti-CTLA4 antibodies or antibody fragments can be used to protect against, prevent or treat cardiovascular diseases, as well as to prevent or inhibit the invasion of viruses such as Lassa virus and Ebola virus, and to treat viral infections.

[0261] In each of the embodiments of the method of treatment described herein, the anti-CTLA4 antibody, antibody fragment, or immunoconjugate of the anti-CTLA4 antibody or antibody fragment may be delivered in a manner consistent with conventional methods associated with 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 of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder. Thus, one aspect of the present invention relates to a method for treating a disease associated with expression of CTLA4, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, antibody fragment, or immunoconjugate of the present invention.

[0262] For administration, anti-CTLA4 antibody, antibody fragment, or immunoconjugate can be formulated as a pharmaceutical composition. Pharmaceutical compositions containing anti-CTLA4 antibody, antibody fragment, or immunoconjugate can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, therapeutic molecules are typically combined with a mixture, solution, or composition that contains a pharma- ceutically acceptable carrier.

[0263] Pharmaceutically acceptable carriers are substances that can be tolerated by recipient patients. 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, etc.

[0264] The form, route of administration, dosage and regimen of pharmaceutical composition will naturally depend on the condition to be 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 to formulate 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 comprises a pharma- ceutically acceptable vehicle for injectable preparations.These may be, in particular, isotonic sterile saline (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts) or may be dry (in particular lyophilized) compositions, which, for example, upon addition of sterile water or saline, allow the constitution of an injectable solution.

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

[0267] Additional excipients include agents that can function as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) as well as agents that prevent denaturation or adhesion to container walls. 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-initose, myonitose, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol), These may include sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-mothioglycerol, 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).

[0268] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to aid in solubilizing the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic 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 (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0270] The dose 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 duration of treatment. To prepare a pharmaceutical composition, an effective amount of the antibody or antibody fragment can be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0271] Suitable dosage forms for injection include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0272] The solution of the active compound as free base or pharmacologically acceptable salt can be prepared in water suitably mixed with surfactant.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and their mixture, and in oil.Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0273] Anti-CTLA4 antibodies or antibody fragments can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and 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), and the like.

[0274] The carrier may also be a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using an agent that delays absorption, for example, aluminum monostearate and gelatin, in the composition.

[0275] Sterile injection solution is prepared by incorporating the required amount of active compound into a suitable solvent, which may contain one or more other ingredients than those listed above, as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle, which contains a basic dispersion medium and other ingredients required from those listed above.For the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized by filtration.

[0276] Preparation of larger volumes or more concentrated solutions for direct injection is also contemplated, and it is envisioned that the use of dimethyl sulfoxide (DMSO) as a solvent will result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

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

[0278] For parenteral administration in aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion solution or injected into the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation 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] The antibody or antibody fragment may be formulated into a therapeutic mixture to deliver about 0.0001-10.0 mg, or about 0.001-5 mg, or about 0.001-1 mg, or about 0.001-0.1 mg, or about 0.1-1.0, or even about 10 mg per dose, or multiple doses at selected time intervals.

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

[0281] In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies or antibody fragments into host cells is contemplated. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0282] Nanocapsules can generally entrap compounds in a stable and reproducible manner.To avoid side effects caused by 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 easily produced.

[0283] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 Å that contain aqueous solution within their core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0284] Pharmaceutical formulations comprising the anti-CTLA4 antibodies or antibody fragments described herein are prepared by mixing such antibodies or antibody fragments having the desired purity, in the form of a lyophilized formulation or aqueous solution, with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers (such as phosphate, citric acid, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride), hexamethonium chloride, benzalkonium chloride, benzethonium 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 (approximately 1 0 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 dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (such as polyethylene glycol (PEG)).

[0285] Exemplary pharma- ceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[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 formulations including histidine-acetate buffers.

[0287] The formulations herein may also contain two or more active ingredients, as necessary, for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other may be combined in a single formulation. For example, in addition to the anti-CTLA4 antibody, antibody fragment or immunoconjugate 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 platinum agent). Such active ingredients are suitably present in combination in amounts 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, which has binding affinity for both CTLA4 and other antigens, thus forming a multispecific (e.g., bispecific) antibody.

[0289] The active ingredient may be encapsulated in microcapsules, which are prepared, for example, by coacervation technology or by interfacial polymerization.For example, hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules may be used in colloid drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively.Such technology is 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 semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices can be in the form of shaped articles, e.g., films, or microcapsules.

[0291] Formulations to be used for in vivo administration are generally sterile. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0292] E. Therapeutic Methods and Compositions Any of the anti-CTLA4 antibodies or antibody fragments provided herein can be used in a method of treatment. In one aspect, an anti-CTLA4 antibody or antibody fragment is provided for use as a medicament. In a further aspect, an anti-CTLA4 antibody or antibody fragment is provided for use in treating 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). In a particular embodiment, an anti-CTLA4 antibody or antibody fragment is provided for use in a method of treatment. In a particular embodiment, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual with cancer, comprising administering to the individual an effective amount of an anti-CTLA4 antibody or antibody fragment. In certain embodiments, the invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having 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 comprising administering to the individual an effective amount of an anti-CTLA4 antibody or antibody fragment. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). In a further embodiment, the invention provides an anti-CTLA4 antibody or antibody fragment for use in inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or tumor stromal function.

[0293] In certain embodiments, the 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., from tumor-associated macrophages), tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or tumor stromal function in an individual, comprising administering to the individual an effective amount of an anti-CTLA4 antibody or antibody fragment to inhibit angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature development (e.g., intratumor or tumor-associated vasculature), 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 for treating cancer, comprising administering an effective amount of the medicament to an individual having cancer. In a further embodiment, the medicament is a medicament for use in a method for treating immune disorders (e.g., autoimmune disorders), cardiovascular disorders (e.g., atherosclerosis, hypertension, thrombosis), infectious diseases (e.g., Ebola virus, Marburg virus) or diabetes, comprising administering an effective amount of an anti-CTLA4 antibody or antibody fragment to an individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). In a further embodiment, the agent is an agent for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or tumor stromal function. In a further embodiment, the agent is an agent for use in a method of inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or tumor stromal function in an individual, comprising administering an effective amount of the agent to the individual to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine release (e.g., from tumor-associated macrophages), inhibit development of tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or inhibit tumor stromal function. An "individual" according to any of the above embodiments may be a human.

[0295] In a further aspect, the 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 one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). An "individual" according to any of the above embodiments may be a human.

[0296] In a further aspect, the 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 one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., as described below). An "individual" according to any of the above embodiments may be a human.

[0297] In a further aspect, the invention provides a method for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature (e.g., from intratumoral or tumor-associated vasculature), 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 secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibit development of tumor vasculature (e.g., from intratumoral or tumor-associated vasculature), and / or inhibit tumor stromal function. In one embodiment, the "individual" is a human.

[0298] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-CTLA4 antibodies or antibody fragments provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-CTLA4 antibodies or antibody fragments provided herein and a pharma- ceutically 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, e.g., as described below.

[0299] In each and every treatment described above, the antibody or antibody fragment of the present invention can be used alone, as an immunoconjugate, or in combination with other agents in the 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, e.g., 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 cytostatic agent. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0300] Such combination therapy as described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations), as well as separate administration, where administration of the antibody or antibody fragment may occur prior to, concurrently with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment may also be combined with radiation therapy.

[0301] Anti-CTLA4 antibodies or antibody fragments may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to physicians. The antibody or antibody fragment is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0302] For the prevention or treatment of disease, the appropriate dosage of the antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being 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 suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg of antibody or antibody fragment / kg patient's body weight to 40 mg of antibody or antibody fragment / kg patient's body weight may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. One typical daily dosage may range from about 1 μg of antibody or antibody fragment / kg patient's body weight to 100 mg of antibody or antibody fragment / kg patient's body weight or more, depending on the factors mentioned above. In the case of repeated administration over several days or more, depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., the patient receives from about 2 to about 20, or, for example, about 6 doses of the antibody or antibody fragment). An initial higher loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0303] Specific dosages of the anti-CTLA4 antibodies or antibody fragments of the invention that may be administered for the prevention or treatment of a disease of interest may be about 0.3, 0.6, 1.2, 18, 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 specific embodiments, dosages may range from 0.3-2.4, 2.4-4.2, 4.2-6.0, 6.0-7.8, 7.8-10.2, 10.2-12, 12-14, 14-16, 16-18, or 18-20 mg of antibody or antibody fragment / kg of patient body weight. Dosages of the antibodies or antibody fragments remain the same 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. Furthermore, the polypeptide having anti-CTLA4 activity is administered in the same amount as the antibody or antibody fragment.

[0304] A single dose of the pharmaceutical formulation of the invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the invention from about 45 μg of antibody or antibody fragment, from about 13,600 mg to about 45 μg of antibody or antibody fragment, or from about 5440 mg to about 45 μg of antibody or antibody fragment. In some embodiments, a single dose of the pharmaceutical formulation of the invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the invention from 135 mg to 1,387 mg, or 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg, etc. In certain embodiments, the amount of the anti-CTLA4 antibody or antibody fragment of the present invention in a single dose of the pharmaceutical formulation 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 the antibody or antibody fragment in a single dose of the pharmaceutical formulation 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. Furthermore, the polypeptide having anti-CTLA4 activity will be included in a single dose of the pharmaceutical formulation in the same amount as the antibody or antibody fragment.

[0305] In one example, an anti-CTLA4 antibody or antibody fragment may be conjugated to another immune checkpoint inhibitor molecule or may 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 an anti-CTLA4 antibody or antibody fragment disclosed in the present application and another immune checkpoint inhibitor molecule administered as separate molecules or as a bispecific antibody. Such a bispecific antibody has a binding activity to CTLA4 and a second binding activity to another immune checkpoint.

[0307] The immune checkpoint may 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). Additional 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 will be understood that any of the above formulations or methods of treatment may be practiced using an antibody fragment or immunoconjugate of the invention in place of, or in addition to, an anti-CTLA4 antibody.

[0310] Enhancing the host's immune function to fight tumors is a subject of growing interest. Traditional methods include (i) enhancing APCs, such as (a) injecting DNA encoding foreign MHC alloantigens into tumors, or (b) transfecting biopsied tumor cells with genes that increase the probability of immune antigen recognition of the tumor (e.g., immune stimulating cytokines, GM-CSF, costimulatory molecules B7.1, B7.2); (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, for example, through stimulation with IL-2 or tumor or both. In addition, isolated T cells that are dysfunctional can also be activated by in vitro application of the anti-PD-L1 antibodies of the invention. Such activated T cells may then be readministered to the host. One or more of these methods can be used in combination with administration of the antibodies, antibody fragments, or immunoconjugates of the invention.

[0311] Traditional therapies for cancer include: (i) radiotherapy (e.g., radiotherapy, x-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiotherapy can be administered via external beam radiation therapy (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., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancing the host's immune response (e.g., vaccines); (v) hormone therapy, or hormone blockade (e.g., when the tumor is hormone sensitive); (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth, and (vii) palliative care, or treatments 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 allow for a more aggressive treatment regimen.

[0312] In the treatment of cancer, any of the above-mentioned conventional treatments for the treatment of cancer immunity can be administered before, after, or simultaneously with the administration of anti-CTLA4 antibody or antibody fragment.In addition, anti-CTLA4 antibody or antibody fragment can be administered before, after, or simultaneously with conventional cancer treatments such as administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or administration of chemotherapeutic agents.

[0313] F. Products and Kits In another aspect of the invention, an article of manufacture is provided that includes an anti-CTLA4 antibody or antibody fragment and other materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture includes a container and a label or package insert on 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 a variety of materials, such as glass or plastic. The container holds the composition, either 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 with 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 to treat the selected condition. Additionally, the article of manufacture may include (a) a first container, in which the composition is contained, the composition comprising an antibody or antibody fragment, and (b) a second container, in which the composition is contained, the composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition.Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution.It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0314] It is to be understood that any of the above products may contain an 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 of the antibodies or antibody fragments of the present invention. The kit comprising the polypeptide, antibody or antibody fragment, or antibody drug conjugate of the present invention is useful for detecting CTLA4 expression (increase or decrease), or for therapeutic or diagnostic assay. The kit 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). A kit may be provided that comprises an antibody for detecting and quantifying CTLA4 in vitro (e.g., ELISA or Western blot). Such an antibody useful for detection may be provided with a label, such as a fluorescent label or a radiolabel.

[0316] The kits further include instructions for their use. In some embodiments, the instructions include instructions required by the U.S. Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kits further include 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 kits include one or more antibodies or antibody fragments. In other embodiments, the kits further include one or more enzymes, enzyme inhibitors, or enzyme activators. In still other embodiments, the kits further include one or more chromatography compounds. In still other embodiments, the kits further include one or more compounds used to prepare the sample for spectroscopic assays. In further embodiments, the kits further include a comparative reference material for interpreting the presence or absence of CTLA4 according to the intensity, color spectrum, or other physical attributes of the indicator.

[0317] The following examples are illustrative of the soft gelatin capsules of the present disclosure, but are not limited thereto. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in the art and obvious to those skilled in the art are within the scope of the present disclosure. EXAMPLES

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

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

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

[0321] Table 3-4 shows the EC binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 6.0 and pH 7.4. 50Values ​​are shown and binding curves for a representative experiment are shown in Figures 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 reduced binding activity at pH 7.4 compared to ipilimumab and ipilimumab analogs. [Table 3] [Table 4]

[0322] In addition, the binding activity of BA-087-05-19 and BA-087-08-32 to immobilized recombinant cynomolgus CTLA4 extracellular domain was also determined by ELISA. Table 5 shows the EC20 binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus CTLA4 at pH 6.0. 50 4A-4B show the binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus monkey CTLA4 at pH 6.0 and pH 7.4. [Table 5]

[0323] EC20 binding activity of BA-087-05-19 and BA-087-08-32 at pH 6.0 in the tumor microenvironment measured by ELISA 50 were found to be 8.18 ng / mL and 9.78 ng / mL for human CTLA4, respectively, and the EC determined for ipilimumab and ipilimumab analogs. 50BA-087-05-19 has similar binding activity to both human and cynomolgus CTLA4 at pH 6.0, whereas BA-087-08-32 has reduced binding activity to cynomolgus CTLA4 at pH 6.0 compared to human CTLA4. The reduced binding activity of BA-087-08-32 to cynomolgus CTLA4 at pH 6.0 seen by ELISA appears to be specific to the ELISA assay, as the same reduction was not observed using either SPR or FACS. The binding activity of BA-087-05-19 and BA-087-08-32 to human or cynomolgus CTLA4 at normal physiological pH 7.4 measured by ELISA was significantly lower than the binding activity at pH 6.0.

[0324] Example 3: pH-dependent binding activity of anti-CTLA4 antibodies The binding activity of the antibodies to CTLA4 was tested using an ELISA assay in a range of pH from 5.0 to 7.4. Recombinant human CTLA4 extracellular domain was immobilized in wells in a range of pH buffers (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 activity was measured using ELISA. Antibodies BA-087-05-19 and BA-087-08-32 were serially diluted and their binding activity to recombinant human CTLA4 extracellular domain was measured. The amount of bound antibodies BA-087-05-19 and BA-087-08-32 was quantified using an anti-human IgG antibody conjugated to horseradish peroxidase (HRP), and then reacted with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to generate 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.

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

[0326] The average OD values ​​(from two replicates) at different pH were plotted against the pH of the buffer using Softmax Pro software (Molecular Devices). Curve fitting was performed using a four-parameter model built into the software. The inflection point of the pH curve (=50% of binding activity at pH 6.0) is equal to parameter C in 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 in Softmax Pro software.

[0327] The average pH for 50% and 90% activity of BA-087-05-19 and BA-087-08-32 was 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 pH for 50% and 90% activity of BA-087-05-19 and BA-087-08-32 determined from the data in experiment 5 were similar to the average pH values ​​calculated using the pH values ​​in experiments 1-4. See Table 6. [Table 6]

[0328] Figure 5 shows the binding activity of BA-087-05-019 and BA-087-08-32, as well as positive control ipilimumab and ipilimumab analogues, to recombinant human CTLA4 in various pH buffers. 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. 90% of the binding activity for BA-087-05-19 and BA-087-08-32 was present at pH 6.34 and pH 6.2, respectively. In addition, weaker binding activity was 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 CTLA4 at pH 6.0 and pH 7.4. CTLA4 extracellular domain (human or cynomolgus) was immobilized on the surface of a sensor chip. Different concentrations of BA-087-05-19 and BA-087-08-32 were injected and the binding interaction with immobilized CTLA4 and control surfaces was monitored in real time. The binding kinetics was calculated using a 1:1 Longmuir model built into the analysis software.

[0330] Antibody BA-087-05-19 exhibited subnanomolar binding activity at pH 6.0. Binding activity decreased approximately two-fold from pH 6.0 to pH 7.4 (K D [pH 6.0] = 0.5 nM, K D [pH 7.4]=1.1 nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 only reached about 20% of the signal level detected at pH 6.0, indicating that at pH 7.4, only a small portion of the BA-087-05-19 present was able to bind to human CTLA4. See Table 7. [Table 7]

[0331] BA-087-08-32 also showed subnanomolar binding activity at pH 6.0. The binding activity decreased approximately 100-fold from pH 6.0 to pH 7.4 (K D [pH 6.0] = 0.45 nM, K D [pH 7.4] = 45 nM). In addition to the lower binding activity at pH 7.4, the SPR signal at pH 7.4 only reaches about 10% of the signal level at pH 6.0, indicating that only a small fraction of the BA-087-08-32 present is capable of binding to human CTLA4 at pH 7.4. See Table 8. [Table 8]

[0332] The commercially available anti-CTLA4 antibody ipilimumab (Yervoy™) was used as a control under the same conditions, and binding activity was found to be very similar at pH 6.0 and pH 7.4 (K D [pH 6.0] = 1.39 nM, K D [pH 7.4] = 1.37 nM). Thus, the binding activity of ipilimumab was independent of pH. See Table 9. Also, the SPR signals obtained are also very similar at both pH 6.0 and pH 7.4. [Table 9]

[0333] The binding activity of antibodies BA-087-05-19, BA-087-08-32, and ipilimumab to cynomolgus monkey CTLA4 was tested using the same conditions as those described above for human CTLA4. All three antibodies have fast off rates and the SPR signal reaches an equilibrium at all antibody concentrations tested. D was calculated by plotting the maximum SPR signal at each concentration against the antibody concentration. At each pH, ​​experiments were performed in triplicate.

[0334] Antibody BA-087-05-19 has a K of 1.96 nM at pH 6.0. D At pH 7.4, the calculated K D Antibody BA-087-08-32 has a K of 5.95 nM at pH 6.0. D At pH 7.4, the resulting SPR signal is D The antibody ipilimumab has a K of 6.58 nM at pH 6.0. D、 and a K of 6.80 nM at pH 7.4. D Binds to cynomolgus monkey CTLA4.

[0335] Example 5: FACS analysis of anti-CTLA4 antibodies The binding activity of antibodies BA-087-05-19 and BA-087-08-32 to human and cynomolgus CTLA4 expressed on the cell surface of CHO cells in pH 6.0 or pH 7.4 buffer was 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 or cynomolgus CTLA4. The amount of antibody bound on the cells was quantified using an anti-human IgG antibody conjugated to a fluorophore. EC at pH 6.0 and 7.4 for binding to cells 50 Values ​​were calculated using a non-linear fitting (variable slope, 4 parameter) model implemented in GraphPad Prism software (version 7.03). Expression levels of human CTLA4 or cynomolgus CTLA4 on the surface of CHO cells were determined using the BD QuantiBRITE™ PE kit.

[0336] At least two independent duplicate FACS experiments were performed for each antibody using each of the cell lines. 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 activity at different concentrations of the antibodies is plotted in these figures.

[0337] The binding activity of BA-087-05-19 and BA-087-08-32 at pH 6.0 as measured by FAC was a mean EC50 of 350.1 and 243.4 ng / mL for human CTLA4, respectively, 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 as measured by FAC were found to have mean EC values ​​of 341.1 ng / mL and 325.4 ng / mL for human CTLA4, respectively, and 337.5 ng / mL and 319.6 ng / mL for cynomolgus CTLA4, respectively. 50 Even at the highest concentration tested, the binding activity of BA-087-05-19 and BA-087-08-32 at pH 7.4 was weaker than that at pH 6.0.

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

[0339] Finally, the saturation of antibodies on CHO cells expressing human or cynomolgus 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 bound less to CHO cells than did the control ipilimumab analog.

[0340] Example 6: ELISA and FACS analysis of anti-CTLA4 antibody stability The binding activity of antibodies BA-087-05-19 and BA-087-08-32 to human CTLA4 was measured using different buffers at pH 6.0 in buffer and at pH 7.4 in buffer. Both ELISA and FACS analysis were used to measure the binding activity. For ELISA analysis, serially diluted BA-087-05-19 and BA-087-08-32 samples were added to wells of the respective buffer that were 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 ELISA data in Figures 9A-9F. EC of binding to human CTLA4 50 Values ​​(ng / mL) were determined by absorbance at 450 nm versus antibody concentration using a Prism variable slope, four-parameter dose-response curve, which was calculated using a nonlinear fitting (variable slope, four parameters) model built into GraphPad Prism software (version 7.03). Tables 10-11 show the EC values ​​for binding to human CTLA4 measured by ELISA in different buffers. 50 Values ​​are shown. Buffers tested included His buffer (His), Tris buffer (Tris), Glutamine buffer (Glu), and no buffer. [Table 10] [Table 11]

[0341] For 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. Binding activity measured by FACS is shown in Figures 10A-10F. EC for binding to human CTLA4 on cells 50 Values ​​(ng / mL) were determined by MFI of the singlet population versus antibody concentration using a Prism variable slope, four-parameter dose-response curve, which was calculated using a nonlinear fitting (variable slope, four parameters) model built into GraphPad Prism software (version 7.03). EC 50 The values ​​are shown in Tables 12-13. [Table 12] [Table 13]

[0342] Example 7: In silico immunogenicity analysis of BA-087-05-19 This study used the EpiVax in silico immunogenicity screening toolkit to determine the potential immunogenicity of BA-087-05-19. The software was accessed through ISPRI, a web-based interactive screening and protein redesign interface. Using the BA-087-05-19 variable domains as input, the software was used to assess 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 show that BA-087-05-19 has a Tregitope-adjusted EpiMatrix protein score of 27.70 and a predicted T-dependent antibody response of 1.29%. This predicted low immunogenicity falls into the group of optimal antibodies (with low effector and high Tregitope content).

[0344] Example 8: Functional assay of enhanced IL-2 secretion by anti-CTLA4 antibodies In this example, the functional activity of antibodies BA-087-05-19 and BA-087-08-32 in inducing the secretion of IL-2 by human lymphocytes stimulated with Staphylococcal Enterotoxin B (SEB) was determined. Serial dilutions of BA-087-05-19, BA-087-08-32, ipilimumab and ipilimumab analogs were added to human peripheral blood mononuclear cells (PBMCs) from normal healthy donors stimulated with SEB. The ability of the antibodies to enhance the secretion of IL-2 in human PBMCs stimulated with SEB 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 above that observed with the addition of isotype control at levels observed with ipilimumab and ipilimumab analogs at pH 6.2. Meanwhile, 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 a mean 1.4-fold increase and BA-087-08-032 promoted a mean 1.5-fold increase in IL-2 production compared to the isotype control at pH 6.2, similar to the increases observed with ipilimumab and ipilimumab analogs. These results indicate that the functional activity of BA-087-05-19 and BA-087-08-32 is comparable to that observed with ipilimumab and ipilimumab analogs at pH 6.2.

[0347] Example 9: Promega® CTLA4 Blocking Assay for Anti-CTLA4 Antibodies The activity of antibodies BA-087-05-19 and BA-087-08-32 in blocking the interaction between human CTLA4 and its ligand (CD80nadCD87) was determined by using an in vitro Promega® CTLA4 blocking assay. Serially diluted BA-087-05-19, BA-087-08-32, ipilimumab and ipilimumab analogs were added to Jurkat effector cells followed by aAPC / Raji cells according to the vendor's protocol. Blockade of the interaction between CTLA4 and its ligand resulted in altered IL-2 pathway activation in Jurkat effector cells, which was quantified using the Bio-Glo® Luciferase Assay Kit.

[0348] As shown in Figure 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 observed with ipilimumab and ipilimumab analogs at pH 6.0. In contrast, as shown in Figure 12B, BA-087-05-19 and BA-087-08-32 were less effective at disrupting the interaction between CTLA4 and its ligands at pH 7.4. These results indicate that the in vitro functional activity of BA-087-05-19 and BA-087-08-32 is comparable to that observed with ipilimumab and ipilimumab analogs at pH 6.0, and is significantly lower than that of ipilimumab and ipilimumab when blocking at pH 7.4.

[0349] Example 10: FACS assay for ligand blockade by anti-CTLA4 antibodies The activity of BA-087-05-19 and BA-087-08-32 to inhibit the interaction of human CTLA4 with its ligands hB7-1 (hCD80) and hB7-2 (hCD86) was assayed by FACS to assess competitive binding at fixed concentrations of BA-087-05-19 and BA-087-08-32 to CHO cells expressing human CTLA4 in the presence of different concentrations of hB7-1 and hB7-2. The amount of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4 cells was quantified using an anti-human IgG antibody conjugated to a fluorophore. As shown in Figures 13A-B, 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 of huCTLA4 with 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 block the interaction between human CTLA4 and its ligands hB7-1 (hCD80) and hB7-2 (hCD86) as efficiently as ipilimumab and ipilimumab analogs. BA-087-05-19 and BA-087-08-32 were only subjected to competitive FACS analysis at pH 6.0 due to very limited binding at pH 7.4.

[0352] Methods used in the examples The ELISA assay was performed using the following protocol: 1) Coat ELISA plates with recombinant CTLA4 antigen at 0.5 μg / mL (06_20_17 and 06_28_17 experiments) or 1 μg / mL (07_06_17 and 07_11_17 experiments) in 100 μL of carbonate-bicarbonate coating buffer. 2) Cover the plate with sealing film and incubate at 4°C overnight. 3) Decant the plate and tap it against a stack of 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 aspirating the contents completely. 5) Add 200 μL of various pH incubation buffers to the wells according to the sample map. Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and tap it against a stack of paper towels to remove any remaining liquid. 7) Serially dilute test substances to 250 ng / mL, 100 ng / mL, or 25 ng / mL in various pH incubation buffers. 8) Add 100 μL / well of diluted test substances to the plate according to the sample map. 9) Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and tap it against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of various pH wash buffers into the wells according to the sample map and completely aspirating the contents. 12) Dilute the HRP secondary antibody 1:2500 in various pH incubation buffers 13) Add 100 μL of HRP secondary antibody diluted in various pH incubation buffers according to the sample map to each well. 14) Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and tap it against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of various pH wash buffers into the wells according to the sample map and completely aspirating the contents. 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 per well of 1N HCl 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 pH were plotted against the pH of the buffer. Curve fitting was performed using a four-parameter model built into the software. The inflection point of the pH curve (50% binding activity) is equal to parameter C in 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 in Softmax Pro software.

[0353] Surface plasmon resonance (SPR) assays were performed using the following protocol: The SPR2 / 4 instrument, SPR affinity sensor (Amine Flat), and immobilization buffer kit are manufactured by Sirra Sensors. The SPR sensor has four flow cells (FC1-FC4), which 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 surface).

[0354] Fixation was performed according to the vendor's recommended protocol. (1) The activator was prepared by mixing 200 mM EDC and 50 mM NHS (Sierra Sensors) immediately prior to injection. The amine sensor chip was activated with the mixture for 480 s at a flow rate of 25 μL / min. (2) Human CTLA4 at 25 μg / mL in 10 mM NaAc (pH 5.0) was injected into FC2 and FC4, respectively, at a flow rate of 25 μL / min for 480 s. The chip surface was deactivated with 1 M ethanolamine-HCl (Sierra Sensors) through FC1-4 at a flow rate of 25 μL / min for 480 s. (3) Control surfaces were activated and deactivated using the same conditions, but without protein injection. (4) Before injecting the analytes, the running buffer was switched to PBST at the required pH. The instrument was equilibrated with the running buffer for 1 h before injecting the first analyte. (5) All analyte injections were performed at 25 μL / min and 25°C.

[0355] BA-087-05-19 was diluted in running buffer (PBST buffer, pH 6.0 or 7.4) 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). BA-087-08-32 was diluted in running buffer (PBST buffer, pH 6.0 or 7.4) 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).

[0356] 100 μL of 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 s association phase followed by a 360 s dissociation phase. Six cycles of analyte runs were repeated with increasing concentrations of analyte. After each cycle of interaction analysis, the chip surface was regenerated by injecting 6 μL of 10 mM glycine (pH 2.0). Each set was run a total of three times at the same pH.

[0357] Flow cell 1 (or 3) without immobilized protein was used as a control surface for reference subtraction. In addition, data with buffer only as analyte (0 nM analyte) was subtracted from each run. The doubly subtracted data was fitted using a 1:1 binding model with the provided analysis software Analyzer R2 (Sierra Sensors). Molar concentrations of analytes were calculated using a molecular weight of 146 kDa.

[0358] Fluorescence-activated cell sorting (FACS) assays were performed using the following protocol.

[0359] Cell staining to determine surface expression of human CTLA4 or cynomolgus monkey CTLA4 1) Transplant 3 x 10 cells into a T-75 flask and culture medium according to the vendor's instructions. 6 Cells are seeded. 2) On the day of FACS analysis, remove and discard the culture medium. 3) The cell layer is briefly rinsed with PBS solution. 4) Add 1.5mL 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) Count the cells using trypan blue staining, then centrifuge at 1500 rpm for 5 minutes at 4°C. 8) Wash the cells once with PBS and resuspend them in 3×10 5 Transfer the 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 on ice at 100 RPM for 1 hour. 10) Wash the cells three 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 on a NovoCyte flow cytometer.

[0360] FACS analysis of CHO cells expressing human CTLA4 or cynomolgus CTLA4 with test antibodies. 1) Harvest the cells (as 3.3, steps 1-7) and wash the cells once with PBS. 2) 3 × 10 cells in FACS buffer, pH 6.0 or pH 7.4 6 Resuspend cells to 375 cells / mL. 3) In a 96-well U-bottom plate, plate 3 x 10 cells in 100 µL of pH 6.0 or pH 7.4 FACS buffer. 5 Aliquot the cells. 4) Spin down the cells and discard the buffer. 5) Test substances are serially diluted in 3-fold dilutions starting at 10 μg / mL (experiments 06-16-17, 06-26-17, and 06-28-17 for a total of 8 data points), or 100 μg / mL (experiment 07_10_17 for a total of 11 data points) in FACS buffer at pH 6.0 or pH 7.4. 6) 100 μL / well of diluted test substances are added to the cells, the wells are mixed gently and incubated 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 pH 6.0 or pH 7.4 wash buffer. 8) Dilute goat anti-human IgG AF488 antibody 1:300 in FACS buffer pH 6.0 or pH 7.4. 9) Add 100 μL of diluted antibody (from step above) to the cells and incubate for 45 minutes on ice, protected from light. 10) Pellet the cells and wash three times with 150 μL of pH 6.0 or pH 7.4 wash buffer. 11) Fix the cells with 4% PFA diluted in 1XPBS for 10 minutes at room temperature, then wash the cells with 1XPBS. 12) Resuspend the cells in 100 μL of 1XPBS. 13) Analyze cells by NovoCyte flow cytometer using Ex 488 nm / Em 530 nm. Collect at least 20,000 cells.

[0361] FACS data were analyzed using a nonlinear fitting (variable slope, four-parameter) model implemented 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 onto dry ice on the bench. Thaw the vial in a 37°C water bath until cells are just thawed (approximately 2 minutes). Visually inspect and gently agitate (do not invert) while thawing. 2) Gently mix the cell suspension in the vial by pipetting up and down 2-3 times and transfer 0.8 mL to the tube labeled "CTLA4 cells" containing 3.2 mL of RPMI + 10% FBS. 3) Spin down the cells at 1500 rpm for 10 minutes and resuspend in 1 mL of RPMI + 10% FBS. Mix well, split the cell suspension into two tubes, spin down the cells, wash the pellet once with either pH 6.0 or pH 7.4 assay medium, and then resuspend the cell pellet in 2 mL of pH 6.0 or pH 7.4 assay medium. 4) Immediately distribute 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 surrounding the sample wells. 6) Starting at 300 μg / mL, serially dilute 3x concentration stocks of test substances in duplicate into pH 6.0 or pH 7.4 assay medium to generate 10x dilution data points. 7) Dispense 25 μL of serially diluted 3x concentrated stock sample material into wells containing 25 μL of CTLA4 Jurkat effector cells according to the layout. 8) Transfer a vial of thawed-ready CTLA4 aAPC / Raji cells (CS186911) from liquid nitrogen storage onto dry ice on the bench. Thaw the vial in a 37°C water bath until the cells are just thawed (approximately 2 minutes). Visually inspect and gently agitate (do not invert) while thawing. 9) Gently mix the cell suspension in the vial by pipetting up and down 2-3 times and transfer 0.8 mL to the tube labeled "aAPC / Raji cells" containing 7.2 mL of RPMI + 10% FBS. 10) Spin down the cells at 1500 rpm for 10 minutes and resuspend in 1 mL of RPMI + 10% FBS. Mix well, split the cell suspension into two tubes, spin down the cells, wash the pellet once with pH 6.0 or pH 7.4 assay medium, and then resuspend the cell pellet in 4 mL of pH 6.0 or pH 7.4 assay medium. 11) Immediately dispense 25 μL of CTLA4 aAPC / Raji cells into the inner 60 wells of the assay plate already containing 50 μL of cell and antibody solution. The assay volume is 75 μL total. 12) Place the lid on the plate and incubate the plate at 37°C in a humidified incubator with 5% CO2 for 16 hours. 13) During the 16 hour induction period, warm the Bio-Glo™ Buffer to ambient temperature using a room temperature water bath before adding the Bio-Glo™ Substrate. 14) Reconstitute the Bio-Glo™ Luciferase Assay System by transferring one bottle of Bio-Glo™ Buffer to the bottle containing 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 to 10 minutes. 18) Luminescence is recorded on a SpectraMax i3X plate reader.

[0363] Although many of the features and advantages of the present invention have been set forth in the foregoing description, together with details of its structure and function, it is to be understood, however, that the disclosure is merely illustrative and that changes in detail may be made, particularly in matters of shape, size and arrangement of parts within the principles of the invention, within the full scope indicated by the broad general meaning of the terms in which the appended claims are expressed.

[0364] All documents mentioned in this specification are incorporated herein by reference in their entirety or provide the disclosure on which they are specifically relied upon. The applicant(s) do not intend to dedicate the disclosed embodiments to the public, and to the extent that the disclosed modifications or variations may not literally fall 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. 1. A pharmaceutical composition for treating a patient with small cell lung cancer, non-small cell lung cancer, renal cancer, liver cancer, melanoma, bladder cancer, cervical cancer, hepatocellular carcinoma, gastric cancer, or renal cancer, comprising an anti-CTLA-4 antibody or antigen-binding antibody fragment, the anti-CTLA-4 antibody or antigen-binding antibody fragment being: A heavy chain variable region comprising three complementarity determining regions having H1, H2, and H3 sequences, (a) the H1 sequence is GFTFSHYTMH (SEQ ID NO:1); (b) the H2 sequence is FIX 1 YX 2 GNX 3 K.X. 4 X 5 AX 6 S.X. 7 KG (SEQ ID NO: 2), (c) the H3 sequence is 8 (SEQ ID NO: 3), In the formula, X 1 is S or D, and X 2 is D, H or I; 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; and A light chain variable region comprising three complementarity determining regions having the sequences L1, L2, and L3, (a) the L1 sequence is 9 SQX 10 X 11 GSSYLA (SEQ ID NO: 4), (b) the L2 sequence is GAFSRATGX 12 (SEQ ID NO:5), (c) the L3 sequence is QQDGSSPWT (SEQ ID NO: 6); In the formula, X 9 is A or I, and X 10 is Y, S or H, and X 11 is V or G, and X 12 is V or I, 13. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises an H2 sequence selected from FIDYHGNNKYYADSVKG, FISYDGNNKIYADSVKG, FISYDGNNKYYADSVKG, FISYDGNYKYYADSVKG, FISYDGNYKYYAKSVKG, FISYHGNNKYEADSVKG, FISYHGNNKYYADSVKG, FISYIGNYKYYADSMKG, and FISYIGNYKYYADSVKG.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises an H3 sequence selected from TGWLGPFDY and TGWLGPFDI.

4. 2. The pharmaceutical composition of claim 1, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises an L1 sequence selected from RASQHVGSSYLA, RASQSVGSSYLA, RASQYGGSSYLA, RASQYVGSSYLA, and RISQYVGSSYLA.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises an L2 sequence selected from GAFSRATGI and GAFSRATGV.

7. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment comprises a light chain variable region having 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.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the anti-CTLA-4 antibody or antigen-binding antibody fragment has a higher binding affinity for CTLA-4 protein at values ​​for conditions in a tumor microenvironment compared to different values ​​for the same conditions occurring in a non-tumor microenvironment.

9. The pharmaceutical composition of claim 8, wherein the condition is pH.

10. 10. The pharmaceutical composition of claim 9, wherein the pH in the tumor microenvironment is in the range of 5.0 to 6.8 and the pH in the non-tumor microenvironment is in the range of 7.0 to 7.

6.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the ratio of the binding affinity of the anti-CTLA-4 antibody or antigen-binding antibody fragment to the CTLA-4 protein at a value of a condition in a tumor microenvironment to a different value of the same condition in a non-tumor microenvironment is at least 1.5:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 20:1, at least 30:1, at least 50:1, at least 70:1, or at least 100:

1.

12. The pharmaceutical composition according to any one of claims 1 to 11, further comprising at least one agent selected from chemotherapeutic agents, radioactive atoms, cytostatic agents and cytotoxic agents.

13. The pharmaceutical composition of claim 12 , wherein the antibody or antibody fragment and the at least one agent are covalently attached to a linker molecule.

14. 14. The pharmaceutical composition of claim 12 or 13, wherein the at least one drug is selected from a maytansinoid, an auristatin, a dolastatin, a calicheamicin, a pyrrolobenzodiazepine, and an anthracycline.

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