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

Abstract

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

Description

[Technical Field]

[0001] This disclosure relates to anti-CTLA4 antibodies, antibody fragments, and immunoconjugates of such antibodies and antibody fragments, as well as the use of antibodies, antibody fragments and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] The vertebrate immune system requires multiple signals to achieve optimal immune activation. See, for example, Janeway, Cold Spring Harbor Symp. Ouant. Biol. 54:1-14 (1989); Paul William E., ed. Raven Press, NY, Fundamental Immunology, 4th edition (1998), particularly pages 12, 13 and 411-478. The interaction between T lymphocytes (T cells) and antigen-presenting cells (APCs) is essential for immune activation. Levels of many aggregated 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 in stimulating antigen-specific T cell proliferation (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 involving cell-cell interactions, particularly interactions between T cells and adjuvant 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)). The immune response is regulated by several T cell surface receptors, including T cell receptor complexes (Weiss, Ann. Rev. Immunol., 4:593-619 (1986)), and other "auxiliary" surface molecules (Allison, Curr. Opin. Immunol., 6:414-419 (1994); Springer (1987), see above). Many of these auxiliary molecules are naturally occurring cell surface differentiation antigens, defined by their reactivity to monoclonal antibodies on the cell surface (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 cytotoxic T cell populations, suggesting that CTLA4 may function in the cytolytic response (Brunet et al. (see above); Brunet et al., Immunol. Rev., 103:21-36 (1988)). Researchers have reported cloning the human counterpart of CTLA4 and mapping it to the same chromosomal region (2d, 33-34) as CD28 (Dariavach et al., Eur: J.Immumol., 18:1901-1905 (1988)), and CD28 (Lafage-Pochitaloff et al., Immunogenetics, 31:198-201 (1990)). Sequence comparison between the DNA of this human CTLA4 and the DNA encoding the CD28 protein revealed remarkable sequence homology, with the greatest degree of homology in the near-membrane 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 co-stimulator (Linsley et al., J.Exp.Med., 176:1595-1604 (1992), Wu et al., J.Exp.Med., 185:1327-1335 (1997), and U.S. Patents No. 5,977,318, 5,968,510, 5,885,796, and 5,885,579). However, others have reported that CTLA4 plays the opposite role as an inhibitor of T cell activation (Krummel, J.Exp.Med., 182:459-465 (1995), Krummel et al., Int'l Immunol., 8:519-523 (1996), Chambers et al., Immunity, 7:885-895 (1997)). CTLA4-deficient mice have been reported to suffer from extensive lymphoproliferation (Chambers et al., see above). Furthermore, it has been reported that CTLA4 blockade enhances the T cell response, worsens antitumor immunity (Leach, Science, 271:1734-1736 (1996)), and exacerbates induced autoimmune diseases (Luhder, J Exp. Med., 187:427-432 (1998)) both in vitro (Walunas et al., Immunity, 1:405-413 (1994)) and in vivo (Kearney, J. Immunol., 155:1032-1036 (1995)). CTLA4 has also been reported to have alternative or additional effects on the early characteristics of the T cell immune response (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. Antibodies blocking CTLA4 may play a pathogenic role in these patients (Matsui, J. Immunol., 162:4328-4335 (1999)).

[0006] CTLA4 has been shown to negatively modulate immune activation through both endogenous and exogenous mechanisms. See Grosso and Kunkel, Cancer Immunity, 13:5 (2013). Specifically, (i) reverse signaling via CD80 and CD86 on APCs suppresses T cell responses and / or promotes the conversion of naive T cells to Tregs; (ii) signaling via CTLA3 stimulates the production of regulatory cytokines such as TGF-β, resulting in inhibition of antigen presentation by APCs and inhibition of T cell function; (iii) when CTLA4 binds to CD80 / CD86, the availability of ligands for binding by CD28 is reduced; (iv) when CTLA4 binds to CD80 / CD86, their transendocytosis (v) CTLA4 causes a decrease in the ability of APCs to activate T cells, by recruiting inhibitory proteins such as PP2A and PTPN11 to T cell synapses and inhibiting CD28 and TCR-mediated signaling, (vi) CTLA4 acts as a high-affinity competitor occupying 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 arrest signals that are important for APC-mediated T cell activation.

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

[0008] Antibodies against CTLA4 have been previously developed. U.S. Patent No. 9,758,583 discloses antibodies or antibody fragments that are said to bind to one or both human and mouse CTLA4, which may be formulated into compositions for the treatment of cancer. Some of the antibodies or antibody fragments are also said to selectively 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 antibody that induced the strongest protection against cancer also induced the fewest 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 relating to or derived from anti-CTLA4 antibodies or antibody fragments. Anti-CTLA4 antibodies and antibody fragments can block human CTLA4 from binding to human B7 and are therefore said to be suitable for the treatment of prostate cancer, kidney cancer, colon cancer, lung cancer, or breast cancer, pathogenic infections, diseases related to the central nervous system, such as amyloidogenic diseases including Alzheimer's disease, as well as diseases having inflammatory or allergic components, such as graft-versus-host disease, host-versus-graft disease, allergies, autoimmune diseases, and other inflammatory diseases.

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

[0012] In one embodiment, the present invention provides isolated heavy chain variable region polypeptides that specifically bind to the CTLA4 protein. These polypeptides comprise three complementarity-determining regions having H1, H2, and H3 sequences. The H1 sequence is GFTFSHYTMH (sequence number 1), The H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (sequence number 2), The H3 sequence is TGWLGPFDX8 (sequence number 3), In the equation, 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 embodiment, the present invention comprises 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 isolated light chain variable region polypeptides comprising three complementarity-determining regions having L1, L2, and L3 sequences, L1 array is RX9SQX 10 X 11 GSSYLA (sequence number 4) The L2 sequence is GAFSRATGX 12 (Sequence ID 5) The L3 sequence is QQDGSSPWT (sequence number 6), In the formula, X9 is A or I, and X 10 However, it is Y, S or H, and X 11 However, it is V or G, and X 12 However, it is either V or I.

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

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

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

[0017] In each of the embodiments described above, 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 embodiment, the present invention provides an anti-CTLA4 antibody or antibody fragment comprising any of the isolated heavy chain variable region polypeptides of the present invention described above.

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

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

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

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

[0025] Each of the aforementioned pharmaceutical compositions may further comprise an immune checkpoint inhibitor molecule different from a polypeptide or antibody or antibody fragment. The immune checkpoint inhibitor molecule may be an antibody or antibody fragment against an immune checkpoint. The immune checkpoint may 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 may 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 WNT proteins. The WNT protein may be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

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

[0028] In yet another embodiment, 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 to 41, and the light chain variable region comprises three complementarity-determining regions having the amino acid sequences of SEQ ID NOs. 42 to 44.

[0029] In the above-described 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 explanation of the drawing]

[0030] [Figure 1] This shows an exemplary sequence alignment of the heavy chain variable region of the anti-CTLA4 antibody of the present invention. [Figure 2] This shows an exemplary sequence alignment of the light chain variable region of the anti-CTLA4 antibody of the present invention. [Figure 3A] This shows a comparison of the binding activity of two of the anti-CTLA4 antibodies of the present invention to human CTLA4 against ipilimumab and ipilimumab analogs (Ipi analogs) at pH 6.0, as measured by enzyme-linked immunosorbent assay (ELISA). [Figure 3B]Figure 3A shows a comparison of the binding activity of the two anti-CTLA4 antibodies of the present invention to human CTLA4 against ipilimumab and ipilimumab analogs (Ipi analogs) at pH 7.4, as measured by ELISA. [Figure 4A] Figure 3A shows a comparison of the binding activity of the two anti-CTLA4 antibodies of the present invention to cynomolgus monkey CTLA4 against ipilimumab and ipilimumab analogs (Ipi analogs) at pH 6.0, as measured by ELISA. [Figure 4B] Figure 3A shows a comparison of the binding activity of the two anti-CTLA4 antibodies of the present invention to cynomolgus monkey CTLA4 against ipilimumab and ipilimumab analogs (Ipi analogs) at pH 7.4, as measured by ELISA. [Figure 5] Figure 3A shows a comparison of the pH-dependent binding activity of the two anti-CTLA4 antibodies of the present invention to human CTLA4 against ipilimumab and ipilimumab analogs (Ipi analogs). [Figure 6A] Figure 3A shows a comparison of the half-maximal effective concentration (EC50) and binding activity of the two anti-CTLA4 antibodies of the present invention to human CTLA4, as measured by fluorescence-activated cell sorting (FACS) using CHO cells at pH 6.0, against ipilimumab and ipilimumab analogs (Ipi analogs). [Figure 6B] Figure 3A shows a comparison of the EC50 and binding activity of the two anti-CTLA4 antibodies of the present invention to human CTLA4, against ipilimumab and ipilimumab analogs (Ipi analogs) at pH 7.4, as measured by FACS using CHO cells. [Figure 7A] Figure 3A shows a comparison of the EC50 and binding activity of the two anti-CTLA4 antibodies of the present invention to cynomolgus monkey CTLA4, as measured by fluorescence-activated cell sorting (FACS) using CHO cells at pH 6.0, against ipilimumab and ipilimumab analogs (Ipi analogs). [Figure 7B]Figure 3A shows a comparison of the EC50 and binding activity of the two anti-CTLA4 antibodies of the present invention to cynomolgus monkey CTLA4, as measured by FACS using CHO cells at pH 7.4, against ipilimumab and ipilimumab analogs (ipi analogs). [Figure 8A] Figure 3A shows a comparison of the EC50 and saturation of human CTLA4 against an ipilimumab analog at pH 7.4, as measured by FACS. [Figure 8B] Figure 3A shows a comparison of the EC50 and saturation of cynomolgus monkey CTLA4 against an ipilimumab analog at pH 7.4, as measured by FACS. [Figures 9A-9F] Figure 3A shows the binding activity of the anti-CTLA4 antibody of the present invention to human CTLA4 at pH 6.0 or pH 7.4, as measured by ELISA, in the presence of different buffers. [Figures 10A-10F] Figure 3A shows the binding activity of the anti-CTLA4 antibody of the present invention to human CTLA4 at pH 6.0 or pH 7.4, as measured by FACS, in the presence of different buffers. [Figure 11A] Figure 3A shows a comparison of the activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), IgG control, and nivolumab (Nivo) analog in blocking IL2 secretion in peripheral blood mononuclear cell (PBMC) culture at pH 6.2. [Figure 11B] Figure 3A shows a comparison of the activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), IgG control, and nivolumab analog in blocking IL2 secretion in PBMC culture at pH 7.4. [Figure 12A] Figure 3A shows a comparison of the EC50 and activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control, which block the interaction between CTLA4 and its ligand at pH 6.0. [Figure 12B] Figure 3A shows a comparison of the EC50 and activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control, which block the interaction between CTLA4 and its ligand at pH 7.4. [Figure 13A] Figure 3A shows a comparison of the EC50 and activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control in competitive binding to human CTLA4, as measured by FACS, as a function of the concentration of the CTLA4 ligand hB7-1 (hCD80). [Figure 13B] Figure 3A shows a comparison of the EC50 and activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control in competitive binding to human CTLA4, as measured by FACS, as a function of the concentration of the CTLA4 ligand hB7-2 (hCD86). [Figure 14A] Figure 3A shows a comparison of the activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control in competitive binding to human CTLA4 at a fixed concentration of the CTLA4 ligand hB7-1, as measured by FACS, as a function of antibody concentration. [Figure 14B] Figure 3A shows a comparison of the activity of the anti-CTLA4 antibody of the present invention against ipilimumab, ipilimumab analog (ipi analog), and IgG control in competitive binding to human CTLA4 at fixed concentrations of the CTLA4 ligand hB7-2, as measured by FACS, as a function of antibody concentration. [Modes for carrying out the invention]

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

[0032] In relation to the measured quantity, the term “approximately” as used herein refers to the normal variation of the measured quantity that would be expected by a person skilled in the art who performs the measurement and exercises due diligence commensurate with the purpose of the measurement and the precision of the measuring instrument used. Unless otherwise indicated, “approximately” refers to a variation of + / - 10% of the given value.

[0033] As used herein, the term “affinity” refers to the sum of the 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 the 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 expressed 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 having one or more modifications in one or more complementarity-determining regions, where such modifications result in improved affinity of the antibody to an antigen compared to a parent antibody without such modifications.

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

[0036] As used herein, the term “antibody” refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules capable of binding to the epitopes of antigens, e.g., Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments, which retain some ability to selectively bind to antigens (e.g., polypeptide antigens) of the antibodies from which they are derived, can be prepared using methods well known in the art (see, for example, Harlow and Lane (above)), and are further described below. Using antibodies, fractional amounts of antigens can be isolated by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or staging of diseases (e.g., tumorigenesis), as well as for therapeutic applications to treat diseases (e.g., tumorigenesis, autoimmune diseases, AIDS, cardiovascular diseases, infections, etc.). Chimeric antibodies, human-like antibodies, humanized antibodies, or fully human antibodies are particularly useful for administration to human patients.

[0037] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and are produced by digesting the entire antibody molecule with the enzyme papain, yielding fragments consisting of intact light and heavy chain portions.

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

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

[0040] An Fv fragment is defined as a genetically modified fragment that contains a variable region of the light chain and a variable region of the heavy chain that is expressed as two separate chains.

[0041] As used herein, the term “antibody fragment” refers to molecules other than intact antibodies, including a portion of an intact antibody that binds to an 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 polyspecific antibodies formed from antibody fragments.

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

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

[0044] As used herein, the terms “cancer” and “malignant” typically refer to or describe a physiological condition in mammals characterized by unregulated cell proliferation / growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, 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, as well as various types of head and neck cancers.

[0045] As used herein, the terms “proliferative disorder” and “proliferative disorder” refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0046] As used herein, the term “chemotherapeutic agent” refers to a chemical substance useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, metsuredopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamamine; and acetogenins (special (including buratacin and buratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacon; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogues topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (so (including synthetic analogs of adzeresin, karzeresin and bizeresin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictin; spongstatin; chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, me Nitrogen mustards such as chloretamine, mechloretamine oxide hydrochloride, melphalan, novembitine, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); oral alpha-4 integrin inhibitor CDP323;Dynemycin containing dynemycin A; esperamicin; and neocardinostatin chromophore and related pigment proteins (endiin antibiotic chromophore), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, 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®), peglirified liposomal doxorubicin (CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and 5-fluorouracil (5-FU); denoph Folic acid analogs such as therine, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine;Demecolsin; Diadicone; Elformitin; Erliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin; Mytansinoids such as Mytansin and Anthamitosin; Mitoguazone; Mitoxantrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (Registered Trademark) Polysaccharide Complex (JHS Natural) Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 Toxin, Beraclin A, Loridine A, Angidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®), Albumin-Modified Nanoparticle Formulation of Paclitaxel (ABRAXANE®), and Docetaxel (TAXOTERE®); Chlorambucil; 6-Thiogunine; Mercaptopurine; Meth Platinum agents such as trexate; cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, which prevents microtubule formation 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 inhibitor RFS2000; difluoromethylornithine (DMF®); and retinoids such as retinoic acid containing bexarotene (TARGRETIN®);Bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog) Antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, such as 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); perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); olafenib, 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 ronafarnib (SCH6636, SARASAR®); and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; two or more combinations of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone);Also mentioned is FOLFOX (an abbreviation for a treatment regimen using oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin).

[0047] Chemotherapy agents as defined herein include “anti-hormone agents” or “endocrine therapeutic agents” that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. These may be hormones themselves, but are not limited to: anti-estrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), doxifen, doroxifen, raloxifene (EVISTA®), trioxyfen, keoxyfen, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (agents that can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestan and exemestane (AROMASIN®), as well as anastrazole (ARIMIDEX®) and letrozole (FEMARA®). This includes nonsteroidal aromatase inhibitors such as aminoglutethimide, as well as other aromatase inhibitors including borozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozol, 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 fluoxymesterone, all-trans lethionic acid, and androgens / retinoids such as fenretinide; onapristone; antiprogesterone; estrogen receptor downregulators (ERD); antiandrogens such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

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

[0049] As used herein, the term “class” of an antibody refers to the type of constant domain or constant region held 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 different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] As used herein, the term “conditionally active antibody” refers to an antibody that is more active under the conditions of the tumor microenvironment compared to the conditions of the non-tumor microenvironment. Conditions in the tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, hypoxia, lower concentrations of glucose, and slightly higher temperature compared to the non-tumor microenvironment. For example, a conditionally active antibody is virtually inactive at normal body temperature but is active at higher temperatures in the tumor microenvironment. In yet another embodiment, a conditionally active antibody is less active in normal oxygenated blood but more active in the hypoxic environment present in a tumor. In yet another embodiment, a conditionally active antibody is less active at the normal physiological pH of 7.2–7.8 but is more active under the acidic pH of 5.8–7.0 or 6.0–6.8 present in the tumor microenvironment. Other conditions known to those skilled in the art exist in the tumor microenvironment and can be used as conditions in this invention, under which anti-CTLA4 antibodies have different binding affinities to CTLA4.

[0051] As used herein, the term “constitutive” refers to the sustained signaling activity of a receptor kinase that does not depend on the presence of a ligand or other activating molecule, as applied, for example, to the activity of CTLA4. Depending on the nature of the receptor kinase, all activity may be constitutive, or the receptor activity may be further activated by the binding of other molecules (e.g., ligands). Cellular events that result in the activation of receptor kinases are well known to those skilled in the art. For example, activation may include oligomerization to higher-order receptor complexes, e.g., dimerization, trimerization, etc. The complex may include a single type of protein, i.e., a homomeric complex. Alternatively, the complex may include at least two different types of proteins, i.e., a heteromeric complex. Complex formation may be caused, for example, by the overexpression of a normal or mutant form of the receptor on the surface of a cell. Complex formation may also be caused by specific mutations or mutations in the receptor.

[0052] As used herein, the term “cell division inhibitor” refers to a compound or composition that stops cell proliferation either in vitro or in vivo. Therefore, cell division inhibitors may significantly reduce the proportion of cells in the S phase. Further examples of cell division inhibitors 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 cell division inhibitor that induces G0 / G1 arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Certain drugs that stop G1, such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and DNA alkylating agents like ara-C, also overflow into S-phase arrest. Further information can be found in Mendelsohn and Israel, eds., "The Molecular Basis of Cancer," Section 1, and Murakami et al., titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WBSaunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel derived from the European yew (TAXOTERE®, Rhone-Poulenc Rorer) is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting the construction of microtubules from tubulin dimers and inhibiting depolymerization, leading to the inhibition of intracellular mitosis.

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

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

[0055] As used herein, the term “detectably labeled” 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. Typical examples of useful detectable labels include, but are not limited to,: molecules or ions that are directly or indirectly detectable based on their light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are detectable by their radioactive properties; and molecules or ions that are detectable by their nuclear magnetic resonance or paramagnetic properties. For example, among the molecules that are indirectly detectable based on light absorption or fluorescence are 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] As used herein, the term “diagnosis” refers to determining susceptibility to a disease or disorder in a subject, determining whether a subject is currently suffering from a disease or disorder, determining the prognosis of a subject suffering from a disease or disorder (e.g., identifying a pre-metastatic or metastatic cancerous state, the stage of cancer, or the cancer’s response to treatment), and determining treatment (e.g., monitoring the subject’s condition to provide information regarding the effectiveness or efficacy of treatment). In some embodiments, the diagnostic methods of the present invention are particularly useful in detecting early-stage cancer.

[0057] As used herein, the term “diagnostic agent” refers to a molecule that can be detected directly or indirectly and used for diagnostic purposes. Diagnostic agents may be administered to a subject or sample. Diagnostic agents may be provided on their own or conjugated to a vehicle such as a conditionally active antibody.

[0058] As used herein, the term “effector function” refers to the biological activity resulting from the Fc region of an antibody, which differs depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated 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 dose” for a drug, for example, a pharmaceutical formulation, refers to an effective amount in terms of the dosage and duration required to achieve the desired therapeutic or prophylactic outcome.

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

[0061] As used herein, the term “framework” or “FR” refers to residues in the variable domain other than those in the hypervariable region (HVR or H1-3 in the heavy chain, and L1-3 in the light chain). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR sequence and FR sequence are V H (or V L In general, they 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 LThis refers to antibodies that include a light chain constant domain (CL) and heavy chain constant domains (CH1, CH2, and CH3). The constant domains may be the constant domains of the natural sequence (e.g., the human natural sequence constant domain) or amino acid sequence variants thereof. Depending on the amino acid sequence of their heavy chain constant domains, full-length antibodies can be assigned to different "classes". There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0063] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced (including the offspring of such cells). Host cells include “transformed organisms” and “transformed cells,” and include primary transformed cells and their offspring, regardless of the number of passages. Offspring may not have nucleic acid content that is exactly identical to that of the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

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

[0065] The "Human Consensus Framework" as used herein refers to the Human Immunoglobulin V L or V HIn the selection of framework sequences, this framework represents the most commonly present amino acid residues. Generally, it is used in human immunoglobulin V. L or V H The sequence is selected from a subgroup of variable domain sequences. Generally, the sequence subgroups are those found 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, V L Regarding this, the subgroup is subgroup κI, as described in Kabat et al. (see above). In one embodiment, V H Regarding this, the subgroup is subgroup III, as described in Kabat et al. (see above).

[0066] As used herein, the term “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human HVR and amino acid residues derived from human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the constant region of an antibody derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0067] As used herein, the terms “hypervariable region” or “HVR” refer to each region of an antibody’s variable domain that is hypervariable in sequence and / or forms a structurally defined loop (“hypervariable loop”). Generally, a natural 4-chain antibody contains six HVRs, and V H There are three in (H1, H2, H3), V LThere are three (L1, L2, L3). HVRs generally contain amino acid residues from a hypervariable loop and / or a "complementarity-determining region" (CDR), the latter of which exhibits the highest sequence variability and / or is 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) are produced at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). H Except for CDR1, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs also contain a "specificity-determining residue" or "SDR," which is a residue that comes into contact with the antigen. The SDR is contained within the region of the CDR and is called an abbreviated CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) arise from amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci., vol.13, pp.1619-1633, 2008). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al. (see above).

[0068] As used herein, the term “immunoconjugate” means an antibody or antibody fragment that is conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radioactive atoms, or cell division inhibitors.

[0069] As used herein, the terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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%, and includes inducing cell death.

[0071] As used herein, the term “isolated” antibody refers to an antibody isolated from its natural environment. In some embodiments, antibodies are purified to a purity of over 95% or over 99%, 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 an overview of antibody purity assessment methods, 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 nucleic acid molecules isolated from components of their natural environment. Isolated nucleic acids typically include nucleic acid molecules found in cells containing nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

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

[0074] As used herein, the term “ligand-independent” refers, for example, to signaling activity that does not depend on the presence of a ligand when applied to receptor signaling activity. Receptors with ligand-independent kinase activity do not necessarily prevent ligand binding to their receptor, leading to further activation of kinase activity.

[0075] As used herein, the term “metastasis” refers to all CTLA4-involved processes that support cancer cells dispersing from a primary tumor, infiltrating lymphatic vessels and / or blood vessels, circulating through the bloodstream, and proliferating in distal lesions (metastases) in normal tissues elsewhere in the body. In particular, it refers to cellular events of tumor cells, such as proliferation, migration, anchorage independence, apoptosis evasion, or secretion of angiogenic factors, that are stimulated or mediated by the non-catalytic or catalytic activity of CTLA4 (preferably including CTLA4 phosphorylation and / or CTLA4-mediated signaling) and that underlie metastasis.

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

[0077] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in that population are identical and / or bind to the same epitope, except for variant antibodies that, for example, contain naturally occurring mutations or may 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 to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is specific to a single determinant on an antigen. Therefore, the modifier “monoclonal” should not be interpreted as indicating an antibody characteristic such as that obtained from a substantially homogeneous population of antibodies, and that it requires the 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 are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0078] As used herein, the term "naked antibody" refers to an antibody that is not conjugated with a heterogeneous moiety (e.g., a cytotoxic moiety) or radiolabeling. Naked antibodies may be present in pharmaceutical formulations.

[0079] As used herein, the term “natural antibody” refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a natural IgG antibody is a heterotetramer glycoprotein with approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (V), also called a variable heavy chain 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 a variable light chain domain or light chain variable domain.L ), followed by the steady light chain (C L The antibody light chain has a domain. Based on the amino acid sequence of its constant domain, the antibody light chain can be assigned to one of two types called kappa (κ) and lambda (λ).

[0080] As used herein, the term “packaging instructions” refers to the instructions that are customarily included in the market packaging of a therapeutic product and include information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings relating to the use of such therapeutic product.

[0081] With respect to the reference polypeptide sequence used herein, the term “percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide sequence, after the sequences have been aligned, gaps introduced where necessary to achieve maximum percent sequence identity, and no conservative substitutions have been considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art of this field using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the percentage value of amino acid sequence identity is generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with 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 source code. The ALIGN-2 program needs to 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 change.

[0082] When ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence B to or with respect to a given amino acid sequence A (or, to be expressed as a given amino acid sequence A that has or contains a specific amino acid sequence identity percentage to or with respect to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y In the formula, X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding section.

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

[0084] As used herein, the term “pharmaceutically acceptable carrier” refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the target. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0085] As used herein, the terms “purified” and “isolated” refer to antibodies or nucleotide sequences according to the present invention, and mean that the indicated molecules are present in the substantial absence of other biomacromolecules of the same kind. As used herein, the term “purified” means preferably that at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of biomacromolecules of the same kind are present. An “isolated” nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide, although the molecule may contain several additional bases or parts that do not adversely affect the basic characteristics of the composition.

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

[0087] As used herein, the term “CTLA4” refers to an immune checkpoint having the amino acid sequence described in U.S. Patents No. 5,434,131, No. 5,844,095, and No. 5,851,795, or any portion or derivative thereof, which recognizes and binds to B7 or interferes with B7 in such a way as to block its binding to CD28 and / or CTLA4 (e.g., endogenous CD28 and / or CTLA4). In certain embodiments, the extracellular domain of wild-type CTLA4 begins with methionine at position +1 and ends with aspartic acid at position +124, or the extracellular domain of wild-type CTLA4 begins with alanine at position -1 and ends with aspartic acid at position +124. Wild-type CTLA4 is a cell surface protein having an N-terminal extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic domain. The extracellular domain binds to a target molecule, such as the B7 molecule. In cells, the naturally occurring wild-type CTLA4 protein is translated as an immature polypeptide containing a signal peptide at its N-terminus. The immature polypeptide undergoes post-translational processing, including cleavage and removal of the signal peptide, to produce a CTLA4 cleavage product with a newly generated N-terminus different from that of the immature form. Those skilled in the art will understand that additional post-translational processing may result from the removal of one or more amino acids from the newly generated N-terminus of the CTLA4 cleavage product. Alternatively, the signal peptide may not be completely removed, resulting in a molecule that starts before methionine, the common starting amino acid. Thus, the mature CTLA4 protein may start with methionine at position +1 or alanine at position -1. The mature form of the CTLA4 molecule includes an extracellular domain or any portion thereof.

[0088] The term "therapeutic dose" of the antibody in this invention means an amount of antibody sufficient to treat the cancer in any medical treatment with a reasonable benefit-risk ratio applicable to that medical treatment. However, it will be understood that the total daily dose of the antibody and composition of this invention is to be determined by the attending physician within the bounds of sound medical judgment. The level of a specific therapeutic dose for any particular patient will depend on a variety of factors, including the disorder being treated and its severity, the activity of the specific antibody used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the timing of administration, route of administration, and excretion rate of the specific antibody used, the duration of treatment, drugs used in combination with or concurrently with the specific antibody used, and similar factors known in the medical field. For example, it is known in the art to start administration of a compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0089] As used herein, the term “single-stranded Fv” (“scFv”) refers to covalently bonded V H ::V L It is a heterodimer, which typically encodes a gene linked by a peptide-encoded linker, V H and V L It is expressed from a gene fusion containing [the specified gene]. "dsFv" is V stabilized by a disulfide bond. H ::V L It is a heterodimer. Divalent and polyvalent antibody fragments can be spontaneously formed by the association of monovalent scFv or generated by ligating monovalent scFv with a peptide linker (e.g., divalent sc(Fv)2).

[0090] The terms “treatment,” “to treat,” or “to treat,” as used herein, refer to a clinical intervention in an attempt to alter the natural course of the individual being treated, which may be carried out either for prevention or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, remission or mitigation of the disease state, and improvement of remission or prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

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

[0092] As used herein, the terms “variable region” or “variable domain” refer to domains in the heavy or light chain of an antibody that are involved in the binding of the antibody to an antigen. (V) H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single V H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, the V from the antibody that binds to the antigen... H or V L Using the domain, antibodies that bind to specific antigens are isolated, and each is a complementary V L or V HYou can screen domain libraries. For example, see Portolano et al., J.Immunol., vol.150, pp.880-887, 1993 and Clarkson et al., Nature, vol.352, pp.624-628, 1991.

[0093] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of nucleic acids to which they are operably ligated. 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. While specific embodiments of the present invention are specifically described herein, those skilled in the art will readily understand that the same principles are equally applicable to and can be used in other systems and methods. Before describing in detail the embodiments disclosed herein, it should be understood that the present invention is not limited in its application to the details of any specific embodiment shown. In addition, the terminology used herein is for illustrative purposes only, not limiting purposes. Furthermore, while certain methods are described by reference to steps presented herein in a particular order, in many cases these steps can be performed in any order as can be understood by those skilled in the art, and therefore novel methods are not limited to a particular arrangement of the steps disclosed herein.

[0095] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Furthermore, 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 used herein and in the claims, such as quantities, molecular weights, percentages, ratios, and reaction conditions, should be understood to be modified by the term "approximately" in all cases, regardless of whether the term "approximately" is present or not. Therefore, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Each numerical parameter should be interpreted, at least in light of the reported number of significant figures and by applying common rounding techniques, not as an attempt to limit the application of the doctrine of equivalents to the claims. Although the numerical ranges and parameters described in the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, each numerical value inherently contains certain errors that inevitably result from the standard deviation found in each test measurement.

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

[0098] Furthermore, each quantity / value or range of each component, compound, substituent, or parameter disclosed herein should be interpreted as being disclosed in combination with any other quantity / value or range of each component / value disclosed herein. Therefore, for the purposes of this description, any combination of quantities / values ​​or ranges of each component / value disclosed herein is also 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, and so on. Furthermore, any specific amount / value of a component, compound, substituent, or parameter disclosed in a description or example 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 the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0100] A. Anti-CTLA4 antibody In one embodiment, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to the human CTLA4 protein. The isolated heavy chain variable region polypeptide comprises three complementarity-determining regions having H1, H2, and H3. The H1 sequence is GFTFSHYTMH (sequence number 1), The H2 sequence is FIX1YX2GNX3KX4X5AX6SX7KG (sequence number 2), The H3 sequence is TGWLGPFDX8 (sequence number 3), In the equation, X1 is S or D, X2 is D, H or I, X3 is N or Y, X4 is Y or I, X5 is Y or E, X6 is D or K, X7 is V or M, and X8 is Y or I.

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

[0102] In another embodiment, the present invention provides an isolated light chain variable region polypeptide that specifically binds to the human CTLA4 protein. The isolated light chain variable region polypeptide comprises three complementarity-determining regions having sequences L1, L2, and L3. L1 array is RX9SQX 10 X 11 GSSYLA (sequence number 4) The L2 sequence is GAFSRATGX 12 (Sequence ID 5) The L3 sequence is QQDGSSPWT (sequence number 6), In the formula, X9 is A or I, and X 10 However, it is Y, S or H, and X 11 However, it is V or G, and X 12 However, it is either V or I.

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

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

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

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

[0107] The present invention includes variants having the heavy-chain variable region and light-chain variable region shown in Figures 1-2, as well as 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-chain variable region and light-chain variable region outside the complementarity-determining region can be mutated according to the principles of substitution, insertion, and deletion discussed in this application. In further embodiments, the constant region can be modified to provide variants.

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

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

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

[0111] Non-conservative substitution involves swapping one member of one of these classes with one of another.

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

[0113] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications can be made in the CDR "hotspots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in the SDR (a-CDR), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and then re-selecting from it 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 one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR-specific approach in which several CDR residues (e.g., 4-6 residues per trial) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0114] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made in the CDR. Such modifications may be in a "hot spot" of the CDR or outside the SDR. Variant V provided above H and V L In certain embodiments of the sequence, each CDR is either unmodified or contains one, two, or three or more amino acid substitutions.

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

[0116] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions (ranging in chain length from 1 residue to polypeptides of 100 or more residues), as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibodies include the fusion of an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody to the N-terminus or C-terminus.

[0117] Modification(s) of the amino acid sequence(s) of the antibodies described herein are intended. 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 CDR inside, simply human antibody V H and V LWhen humanized antibodies are produced by transplanting them into FR, it is known that their antigen-binding activity is reduced compared to the antigen-binding activity of the original antibodies derived from non-human animals. This is true not only for CDR but also for FR, where the V of non-human antibodies H and V L Several amino acid residues are thought to be directly or indirectly related to antigen-binding activity. Therefore, these amino acid residues are considered to be related to the V of human antibodies. H and V L Substitution with different amino acid residues derived from FR would likely reduce binding activity. To solve this problem, in antibodies transplanted with human CDR, the V of human antibodies H and V L Within the amino acid sequence of the FR, it is necessary to attempt to identify amino acid residues that are directly related to antibody binding, amino acid residues that interact with amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to binding to the antigen. The reduced antigen-binding activity can be increased by substituting the identified amino acids with amino acid residues from the original antibody derived from a non-human animal.

[0118] Modifications and alterations are made to the structure and encoding DNA sequence of the antibody of the present invention, and a functional molecule encoding an antibody that still possesses the desired characteristics can be obtained.

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

[0120] Furthermore, a further object of the present invention also includes function-conserving variants of the antibody of the present invention.

[0121] A “function-conserving variant” is one in which a given amino acid residue in a protein or enzyme is altered without altering the overall conformation and function of the polypeptide, and this includes, but is not limited to, amino acid substitutions with amino acids that have similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conserved may differ in the protein, and as a result, the percentage sequence similarity of the protein or amino acids between any two functionally similar proteins may change, for example, 70% to 99% when the similarity is determined according to an alignment scheme such as the clustering method based on the MEGALIGN algorithm. A “function-conserving 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%, when determined by the BLAST or FASTA algorithm, and which has the same or substantially similar properties or functions as the native protein or parent protein being 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 their amino acids are identical, or if 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.) pile-up program, or one of sequence comparison algorithms such as BLAST or FASTA.

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

[0124] In the field of the art, it is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores, and that this still results in proteins with similar biological activity, i.e., proteins that are still equivalent in biological function.

[0125] Therefore, as outlined above, amino acid substitutions are generally based on the relative similarities of the substituents on the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions that take into account the various characteristics mentioned above are known to those skilled in the art and include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, as well as valine, leucine and isoleucine.

[0126] Glycosylated variant In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are generated or removed.

[0127] If an antibody contains an Fc region, the carbohydrate bound to it can be modified. Natural antibodies produced by mammalian cells typically contain branched or bibranched oligosaccharides, generally bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH, vol.15, pp.26-32, 1997. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention can be performed to produce antibody variants with specific improved properties.

[0128] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 for the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546, for example. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of the Fc region residue), although Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S.2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S.2003 / 0115614, U.S.2002 / 0164328, U.S.2004 / 0093621, and U.S.2004 / 01321. References include 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 lacking 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 α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol.87, pp.614-622, 2004; Kanda, Y. et al. Biotechnol. Bioeng., vol.94, pp.680-688, 2006, and WO2003 / 085107).

[0129] Antibody variants containing branched oligosaccharides are also provided, for example, in which the branched oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S.2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0130] Fc region variant In certain embodiments, an Fc region variant can be generated by introducing one or more amino acid modifications into the Fc region of an antibody provided herein. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes one or more amino acid modifications (e.g., α substitutions) at one or more amino acid positions.

[0131] In certain embodiments, the present invention envisions antibody variants possessing some, but not all, effector functions, which are desirable candidates for applications where the in vivo half-life of the antibody is important, but specific effector functions (such as ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while mononuclear cells 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 for evaluating the ADCC activity of a target molecule include U.S. Patent No. 5,500,362 (see also 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, and U.S. Patent No. 5,821,337 (see also Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assays may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, Calif.) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., 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 MJ Glennie, Blood, vol.103, pp.2738-2743, 2004). Furthermore, FcRn binding and in vivo clearance / half-life determination can be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol., vol.18, pp.1759-1769, 2006).

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

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

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

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

[0136] Antibodies that improve binding to the neonatal Fc receptor (FcRn) (Guyer et al., J.Immunol., vol.117, pp.587-593, 1976 and Kim et al., J.Immunol., vol.24, p.249, 1994), which is involved in the transfer of maternal IgG to the fetus, and increase its half-life, are described in US2005 / 0014934. These antibodies contain an Fc region with one or more substitutions, improving the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions in 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 of 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. Patents No. 5,648,260, 5,624,821, and WO94 / 29351.

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

[0138] antibody derivative In certain embodiments, the antibodies or antibody fragments provided herein may be further modified to include additional non-proteinogenic moieties known and readily available in the art. Suitable moieties for derivatization of antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limited 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), as well as dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody or antibody fragment may vary, and if two or more polymers are bound, they may be the same or different molecules. In general, the number and / or types of polymers used in derivatization can be determined, but are not limited, based on considerations including the specific properties or functions of the antibody or antibody fragment to be improved, and whether the derivative will be used therapeutically under given conditions.

[0139] In another embodiment, a conjugate of an antibody or antibody fragment and a non-proteinaceous moiety is provided, which 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 may be of any wavelength, but is not limited, and includes wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells adjacent to the antibody-non-proteinaceous moiety.

[0140] In another embodiment, the present 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, and 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, and 37.

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

[0142] In certain embodiments, the anti-CTLA4 antibody or antibody fragment of the present invention has a concentration of approximately 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 (for example, 10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10 -13 M) has a dissociation constant (Kd) with CTLA4. In one embodiment, the ratio of the Kd of the antibody or antibody fragment with CTLA4 at a value in the tumor microenvironment to the Kd at different values ​​in the same conditions in the non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

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

[0144] According to another embodiment, Kd is measured at approximately 10 response units (RU) at 25°C using a surface plasmon resonance assay with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip. Briefly, according to the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, serially diluted 2-fold dilutions of Fab (0.78 nM to 500 nM) are injected into 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 coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. off / k on Calculate as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The on rate is 10 by the surface plasmon resonance assay described above. 6 M -1 s -1 If it exceeds this, the on rate can be determined while increasing the antigen concentration, as measured by a spectrometer such as an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirring cuvette, using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C.

[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, such as Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragment formed from the antibody fragment, and a polyspecific antibody are used. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody, or another antibody class or isotype as defined herein. For an overview of specific antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For an overview of the scFv fragment, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as WO93 / 16185, and U.S. Patents Nos. 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0146] The diabodies of the present invention may be divalent or bispecific. For example, 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. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat.Med.,vol.9,pp.129-134,2003.

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

[0148] Antibody fragments can be prepared by a variety of techniques, but are not limited to, the protein digestion of intact antibodies, as well as production by recombinant host cells (e.g., Escherichia 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 in Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody is modified compared to the class or subclass of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0150] In certain embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, where the CDR (or a portion thereof) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may also optionally contain at least a portion of the human constant region. In some embodiments, several FR residues of the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the CDR residue is derived) to restore or improve the specificity or affinity of the antibody, for example.

[0151] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further, for example, 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. This is described in al., Methods, vol.36, pp.25-34, 2005 (SDR(a-CDR) grafts), Padlan, Mol.Immunol., vol.28, pp.489-498, 1991 ("resurfacing" is described), Dall'Acqua et al., Methods, vol.36, pp.43-60, 2005 ("FR shuffling" is described), 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 ("guided selection" approach to FR shuffling is described).

[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 specific subgroups of light chain 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 maturation (somatic mutation) 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 (e.g., Baca et al. See also 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 polyspecific antibody, such as a bispecific antibody. A polyspecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is for CTLA4 and the other is for another antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of CTLA4. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing CTLA4. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0154] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol.305, pp.537-540, 1983, WO93 / 08829, and Traunecker et al., EMBO J. vol.10, pp.3655-3659, 1991), and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Furthermore, polyspecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimer molecules (WO2009 / 089004A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, vol. 229, pp. 81-83, 1985), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), or using "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al.) They can also be prepared by using single-stranded Fv(scFv) dimers (see, for example, Gruber et al., J.Immunol., vol.152, pp.5368-5374, 1994), and by preparing tripspecific antibodies (see, for example, Tutt et al., J.Immunol., vol.147, pp.60-69, 1991).

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

[0156] The anti-CTLA4 antibody or antibody fragment of the present invention can be produced using the recombinant method and composition described in detail in US2016 / 0017040.

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

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

[0159] In one embodiment, the immune conjugate is an antibody-drug conjugate (ADC) in which an antibody or antibody fragment is conjugated to one or more drugs, and is not limited to mytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425235B1), auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483) See U.S. Patent Nos. 5,780,588 and 7,498,298), drastatin, calicheamycin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296, Hinman See 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 (see Kratz et al., Current Med. Chem., vol.13, pp.477-523, 2006, Jeffrey et al., Bioorganic & Med. Chem. Letters, vol.16, pp.358-362, 2006, Torgov et al., Bioconj. Chem., vol.16, pp.717-721, 2005, Nagy et al., Proc. Natl. Acad. Sci. USA, vol.97, pp.829-834, 2000, Dubowchik et al.,Bioorg.& Med.Chem.Letters,vol.12,vol.1529-1532,2002, King et al.,J.Med.Chem.,vol.45,pp.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, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0161] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to a radioactive atom, forming a radioconjugate. Various 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 212Examples include radioactive isotopes of , and Lu. When a radioactive conjugate is used for detection, it may also contain a spin label (also known as magnetic resonance imaging, MRI) for nuclear magnetic resonance (NMR) imaging, such as radioactive atoms for scintigraphy studies, e.g., tc99m or I123, or 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 with cytotoxic agents can be prepared 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), difunctional derivatives of imide esters (e.g., dimethylHCl adipimidoate), active esters (e.g., disaxinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine 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 may be a “cleavable linker” that facilitates the release of cytotoxic drugs into cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or linkers containing disulfides may be used (Chari et al., Cancer Res., vol.52, pp.127-131, 1992, U.S. Patent No. 5,208,020).

[0163] The immunoconjugate herein explicitly contemplates, but is not limited to, conjugates prepared with crosslinking reagents including commercially available 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 (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A), as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate).

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

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

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

[0167] In one embodiment, the linker has a functionality that allows it to react with free cysteine ​​present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include activated esters such as maleimide, haloacetamide, α-haloacetyl, and succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. For example, see the conjugation method on page 766 of Klussman, et al, Bioconjugate Chemistry, vol. 15, pp. 765-773, 2004.

[0168] In some embodiments, the linker has a functionality that allows it to react with electrophiles present on the antibody. Exemplary such electrophiles include, but are not limited to, aldehydes and ketone carbonyl groups. In some embodiments, the heteroatoms of the linker's reactive functional group can react with electrophiles on the antibody to form a covalent bond with the antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

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

[0170] The linker may be a “cleavable linker” that facilitates the release of a drug. Non-exclusive exemplary cleavable linkers include acid-unstable linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photo-unstable linkers, or linkers containing disulfides (Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Patent No. 5,208,020).

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

[0172] In some embodiments, the linker component includes an “extension unit” (A) that links the antibody to another linker component or drug moiety. Non-limiting and illustrative extension units are shown below (wavy lines indicate covalent binding sites to the antibody, drug, or additional linker component). [ka]

[0173] In some embodiments, the linker component includes an "amino acid unit" (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating the 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-homolynise (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid units may include naturally occurring amino acid residues and / or trace amino acids and / or unnatural amino acid analogs, such as citrulline amino acid units, which can be designed and optimized for enzymatic cleavage by certain enzymes, e.g., tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

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

[0175] In some embodiments, the linker component includes “spacer units” (Y) that either directly link the antibody to the drug moiety or link it via elongation units and / or amino acid units. The spacer units may be “self-immolative” or “non-self-immolative.” “Non-self-immolative” spacer units are those in which some or all of the spacer unit remains bound to the drug moiety upon cleavage of the ADC. Examples of non-self-immolative spacer units, but not limited to, include glycine spacer units and glycine-glycine spacer units. In some embodiments, enzymatic cleavage of an ADC containing glycine-glycine spacer units by a tumor cell-associated protease results in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety is subjected to a hydrolysis step in tumor cells, so that the glycine-glycine spacer unit is cleaved from the drug moiety.

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

[0177] Other examples of self-destructing 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. Patent No. 7,375,078, Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999), and ortho or para aminobenzyl acetals. In some embodiments, 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 amide (Amsberry et al., J. Org. Chem., vol.55, p.5867, 1990). Drug binding to the α-carbon of a glycine residue is another example of a self-destructing spacer that may be useful in ADCs (Kingsbury et al., J. Med. Chem., vol.27, p.1447, 1984).

[0178] In some embodiments, linker L may be a dendritic linker for covalently attaching two or more drug moieties to the 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 the drug to the antibody (i.e., the load related to the potency of the ADC). Therefore, if the antibody has only one reactive cysteinethiol group, multiple drug moieties can be attached via the dendritic linker.

[0179] In the context of ADCs, a non-restrictive and exemplary linker is given by the following formula I:

Chem.

Chem.

[0180] Further non-limiting exemplary ADCs include the following structures:

Chem.

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

[0182] The compounds of the present invention are not limited to, but it is explicitly intended that ADCs be prepared using the following linker reagents: bis-maleimide-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-vinyl sulfone (HBVS), succinyl Midyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidecaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), succinimil 3-(bromoacetamide)propionate (SBAP), succinimidoiodoacetate (SIA), succindiyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2- Pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl-6-[(beta-maleimidopropionamide)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- The following are included: MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and succinimidyl-(4-vinylsulfone)benzoate (SVSB), and bis-maleimide reagents: dithiobismaleimideethane (DTME), 1,4-bismaleimidebutane (BMB), 1,4-bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidehexane (BMH), bismaleimideethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below);Difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, the bis-maleimide reagent allows the thiol group of the antibody cysteine ​​to be bound to a thiol-containing drug moiety, linker, or linker-drug intermediate. 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 various commercial sources such as Pierce Biotechnology, Inc. (Rockford, Ill.) and Molecular Biosciences Inc. (Boulder, Col.), 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, U.S.2003 / 130189, U.S.2003 / 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 radioactive nucleotides to antibodies. See, for example, WO94 / 11026.

[0185] ii) Exemplary drug portion 1) Mytansin and Mytansinoids In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more mytansinoid molecules. Mytansinoids are derivatives of mytansin and are mitotic inhibitors that act by inhibiting the polymerization of tubulin. Mytansin was initially isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce mytansinoids, such as mytansinol and C-3 mytansinol ester (U.S. Patent No. 4,151,042). Synthetic mytansinoids include, for example, U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, and This information is disclosed in patents No. 4,313,946, No. 4,315,929, No. 4,317,821, No. 4,322,348, No. 4,331,598, No. 4,361,650, No. 4,364,866, No. 4,424,219, No. 4,450,254, No. 4,362,663, and No. 4,371,533.

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

[0187] Certain mytansinoids suitable for use as the mytansinoid drug moiety 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). Mytansinoids may also be prepared synthetically according to known methods.

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

[0189] Exemplary mytansinoid drug moieties may also have the following modifications: C-9-SH (US Patent No. 4,424,219) (e.g., prepared by reaction of mytansinol with H2S or P2S5), C-14-alkoxymethyl (demethoxy / CH2OR) (US Patent No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (US Patent No. 4,450,254) (e.g., prepared from Nocardia), C-15-hydroxy / acyloxy (US Patent No. 4,364,866) (e.g., prepared by conversion of mytansinol by Streptomyces), C-15-methoxy (US Patents No. 4,313,946 and 4,315,929) (e.g., Trewija (Isolated from nudlflora), C-18-N-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (e.g., prepared by demethylation of mytansinol by Streptomyces species), and 4,5-deoxy (U.S. Patent No. 4,371,533) (e.g., prepared by titanium trichloride / LAH reduction of mytansinol).

[0190] Many positions on the mytansinoid compound are useful as bonding sites. For example, ester bonds can be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction may occur at the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified 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 mytansinol or a mytansinol analog.

[0191] The mytansinoid drug portion includes those with the following structures: [ka] Here, the wavy lines indicate the covalent bond of the sulfur atom of the mytansinoid drug moiety to the linker of the ADC. Each R can independently be H or C1-C6 alkyl. The alkylene chain bonding the amide group to the sulfur atom may be methanyl, etanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Patent 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 mytansinoid drug moiety are intended for the ADC of the present invention, i.e., any combination of R and S configurations at the chiral carbon (U.S. Patents 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 mytansinoid drug moiety has the following stereochemistry: [ka]

[0193] Exemplary embodiments of the mytansinoid drug portion include, but are not limited to, DM1, DM3, and DM4 having the following structures: [ka] Here, the wavy line indicates the covalent bond 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 the thiol group of the antibody via a BMPEO linker has the following structure and abbreviations: [ka] In the formula, 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, 1 to 7, 1 to 5, or 1 to 4.

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

[0196] In some embodiments, antibody-mytansinoid conjugates can be prepared by chemically conjugating an antibody to a mytansinoid molecule without significantly reducing the biological activity of either the antibody or the mytansinoid molecule. See, for example, U.S. Patent No. 5,208,020. In some embodiments, ADCs conjugated with an average of 3-4 mytansinoid molecules per antibody molecule have shown efficacy in enhancing the cytotoxicity of target cells without adversely affecting the antibody's function or solubility. In some cases, even a single molecule of toxin / antibody is expected to enhance cytotoxicity more than using a naked antibody.

[0197] Examples of conjugates for creating antibody-mytansinoid conjugates include, for example, the conjugates described herein, and the conjugates described in U.S. Patent No. 5,208,020, EP0425235B1, Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992, U.S. 2005 / 0276812A1, and U.S. 2005 / 016993A1.

[0198] (2) Auristatin and Dorastatin The drug portions include drastatin, auristatin, and their analogs and derivatives (U.S. Patents 5,635,483, 5,780,588, 5,767,237, and 6,124,431). Auristatin is a derivative of drastatin-10, a compound from marine mollusks. While not intended to be bound by any particular theory, drastatin and auristatin 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 (US Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The drastatin / aulistatin drug moiety can bind to antibodies via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO02 / 088172, Doronina et al., Nature Biotechnology, vol.21, pp.778-784, 2003; Francisco et al., Blood, vol.102, pp.1458-1465, 2003).

[0199] An exemplary embodiment of auristatin is the N-terminally linked monomethyl auristatin drug moiety disclosed in U.S. Patent 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 covalent binding sites to the antibody or antibody-linker component, and each site is independent of the others. R 2 These are selected from H and C1-C8 alkyl groups. R 3The elements are selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle). R 4 The elements are selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle). R 5 It is selected from H and methyl, Or, R 4 and R 5 They both form a carbon ring, and the formula is -(CR a R b ) n - has, R a and R b n is independently selected from H, C1-C8 alkyl, and C3-C8 carbon rings, and n is selected from 2, 3, 4, 5, and 6. R 6 These are selected from H and C1-C8 alkyl groups. R 7 The elements are selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle, and C1-C8 alkyl-(C3-C8 heterocycle). Each R 8 These are independently selected from H, OH, C1-C8 alkyl, C3-C8 carbon ring, and O-(C1-C8 alkyl), R 9 These are selected from H and C1-C8 alkyl groups. R 10 It is selected from aryl or C3-C8 heterocycles, Z is O, S, NH, or NR 12 And R 12 These are C1-C8 alkyl groups, 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 ) Selected from 2, m is an integer in the range of 1 to 1000. R 13 These are C2-C8 alkyl groups, R 14 is H or C1-C8 alkyl, e is independently H, COOH, and -(CH2) in each instance. n -N(R 16 )2, -(CH2) n -SO3H, or -(CH2) n -SO3-C1~C8 alkyl, e is independently H, C1-C8 alkyl, or -(CH2) in each instance. n -COOH, R 18 -C(R 8 )2-C(R 8 )2-aryl, -C(R 8 )2-C(R 8 )2-(C3~C8 heteroalgebra), and -C(R 8 )2-C(R 8 )2-(C3~C8 carbon rings) are selected, n is an integer in the range of 0 to 6.

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

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

[0202] In yet another embodiment, R 8 Each occurrence of this value is -OCH3.

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

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

[0205] In one embodiment, R 10 That is Ariel.

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

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

[0208] In one embodiment, if Z is -NH, then R 11 is -CH(R 15 )2, and in the formula, R 15 ha-(CH2) n -N(R 16 )2, R 16 is -C1~C8 alkyl or -(CH2) n -COOH

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

[0210] Formula D E An exemplary embodiment of auristatin is MMAE, where the wavy line indicates covalent binding of the antibody-drug conjugate to linker (L): [ka]

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

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

[0213] Non-limiting and exemplary embodiments of ADCs of Formula I, including MMAF and various linker components, include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates containing MMAF bound to an antibody by a non-proteolytic cleavable linker have been shown to have equivalent activity to immunoconjugates containing MMAF bound to an antibody by a proteolytic cleavable linker (Doronina et al., Bioconjugate Chem., vol.17, pp.114-124, 2006). In some such embodiments, drug release is thought to be affected by antibody degradation in cells.

[0214] Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, by liquid-phase synthesis (see, e.g., E. Schroder and K. Lubke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press). The auristatin / dostatin drug portion may be prepared in some embodiments according to the following methods: U.S. Patent Nos. 7,498,298, 5,635,483, and 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, formula D such as MMAE E The auristatin / dostatin drug portion, and D such as MMAF E Drug-linker intermediates and their derivatives, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, can be prepared using the methods described in U.S. Patent No. 7,498,298, Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006, and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003, and then conjugated to the antibody of interest.

[0216] (3) Calicheamycin In some embodiments, the immunoconjugate comprises an antibody or antibody fragment conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics and their analogues can generate double-strand 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. Therefore, in some embodiments, cellular uptake of these drugs through antibody-mediated internalization can significantly enhance their cytotoxic effects. Non-limiting and exemplary methods for preparing antibody-drug conjugates having a calicheamicin drug moiety are described, for example, in U.S. Patents 5,712,374, 5,714,586, 5,739,116, and 5,767,285.

[0217] (4) Pyrrolobenzodiazepine In some embodiments, the ADC contains pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. PBD, a naturally occurring anthramycin, was first reported in 1965 (Leimgruber et al., J.Am.Chem.Soc., vol.87, pp.5793-5795, 1965; Leimgruber et al., J.Am.Chem.Soc., vol.87, pp.5791-5793, 1965). Subsequently, several PBDs, both natural and analogous, have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994), including dimers of the tricyclic PBD skeleton (U.S. Patents No. 6,884,799, No. 7,049,311, No. 7,067,511, No. 7,265,105, No. 7,511,032, No. 7,528,126, and No. 7,557,099). While not intended to be bound by any particular theory, the dimeric structure is thought to provide a suitable three-dimensional shape for equihelicality with the minor groove of type B DNA, allowing it to fit snugly to the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp.3-11 (1975); Hurley and Needham-VanDevanter, Acc.Chem.Res., vol.19, pp.230-237, 1986). Dimeric PBD compounds having a C2 aryl substituent 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 are conjugated to antibodies, and the resulting ADCs have been shown to possess anticancer properties. Non-limiting and exemplary linking sites on PBD dimers include five-membered pyrrolo rings, tethering between PBD units, and N10-C11 imine groups (WO2009 / 016516, US2009 / 304710, US2010 / 047257, US2009 / 036431, US2011 / 0256157, WO2011 / 130598).

[0219] The non-limiting and exemplary PBD dimer components of ADC are: [ka] or a salt or solvate thereof, in the formula, The wavy line indicates the covalent bonding site to the linker. The dotted line indicates the presence of an optional double bond between C1 and C2, or between C2 and C3. R 2 These are independently H, OH, =O, =CH2, CN, R, OR, =CH-R D , =C(R D )2, selected from O-SO2-R, CO2R, and COR, and optionally further selected from halo or dihalo, R D These are independently selected from R, CO2R, COR, CHO, CO2H, and Halogen. R 6 and R 9 These are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn, and Halo. R 7 These 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 It is either H, R, or SO3M if Q is O, and M is a metal cation. R and R' are each independently and arbitrarily substituted C 1~8 Alkyl, C1~12 Alkyl, C 3~8 Heterocyclyl, C 3~20 Heterogeneous algebras, and C 5~20 Selected from aryl groups and optionally associated with the group NRR', R and R', together with the nitrogen atom to which they are bonded, form optionally substituted 4-membered, 5-membered, 6-membered, or 7-membered heterocycles. R 12 , R 16 , R 19 and R 17 These are R, respectively. 2 , R 6 , R 9 and R 7 As defined, R'' is C3~ 12 It is an alkylene group, and this chain may be interrupted by one or more heteroatoms, e.g., O, S, N(H), NMe, and / or aromatic rings (e.g., benzene or pyridine), and these rings may be optionally substituted. X and X' are independently selected from O, S, and N(H).

[0220] In some embodiments, R and R' are each independently substituted with an optionally replaced C. 1~12 Alkyl, C 3~20 Heterogeneous algebras, and C 5~20 Selected from aryl groups, and optionally associated with the groups NRR', R, and R', they form optionally substituted 4-membered, 5-membered, 6-membered, or 7-membered heterocycles together with the nitrogen atoms to which they are bonded. In some embodiments, R 9 and R 19 H is H. In some embodiments, R 6 and R 16 H is H.

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

[0222] In some embodiments, R 2 and R 12 These are independently selected from H and R. In some embodiments, R 2 and R 12 R is independent of R. In some embodiments, R 2 and R 12 This is an independently and arbitrarily substituted C 5~20 Aryl or C 5~7 Aryl or C 8~10 It is an arrow. In some embodiments, R 2 and R 12 R is independently and optionally substituted with phenyl, thienyl, naptyl, pyridyl, quinolinyl, or isoquinolinyl. In some embodiments, R 2 and R 12 These are independently =O, =CH2, and =CH-R D , and =C(R D ) Selected from 2. In some embodiments, R 2 and R 12 These are, respectively, =CH2. In some embodiments, R 2 and R 12 These are H, respectively. In some embodiments, R 2 and R 12 Each of these is = O. In some embodiments, R 2 and R 12 Each of these is =CF2. In some embodiments, R 2 and / or R 12 These are independently, =C(R D )2. In some embodiments, R 2 and / or R 12 These are independently, =CH-R D That is the case.

[0223] In some embodiments, R 2 and / or R 12 ga = CH-R D If so, each unit may independently have one of the following configurations: [ka] In some embodiments, a=CH-R D This is found in configuration (I). In some embodiments, R'' is a C3 alkylene group or a C5 alkylene group.

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

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

[0226] (5) Anthracyclines In some embodiments, the ADC may contain anthracyclines, which are antibiotic compounds exhibiting cytotoxic activity. While not intended to be bound by any particular theory, studies have shown that anthracyclines may act to kill cells through several different mechanisms, including: 1) inhibiting DNA-dependent nucleic acid synthesis by intercalating the drug molecule into the cell's DNA; 2) drug-induced production of free radicals that then react with cellular macromolecules to cause cell damage; and / or 3) interactions between the drug molecule and the cell membrane (see, e.g., C. Peterson et al., “Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia” in Anthracycline Antibiotics In Cancer Therapy; NRBachur, “Free Radical Damage” (ibid., pp. 97-102)). These potentially cytotoxic anthracyclines have been used to treat numerous 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 and exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and their derivatives. 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.) See al., J. Med. Chem., vol. 45, pp. 4336-4343, 2002, EP0328147, U.S. Patent No. 6,630,579). The antibody-drug conjugate BR96-doxorubicin specifically reacts with the tumor-associated antigen Lewis-Y and has been evaluated in Phase I and Phase II trials (Saleh et al., J. Clin. Oncology, vol. 18, pp. 2282-2292, 2000; Ajani et al., Cancer Jour., vol. 6, pp. 78-81, 2000; Tolcher et al., J. Clin. Oncology, vol. 17, pp. 478-484, 1999).

[0228] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol.11, pp.1608-1617, 2005). Nemorubicin is a semi-synthetic analog of doxorubicin, having a 2-methoxymorpholino group on the glycoside amino of doxorubicin, and has undergone 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 a Phase II / Phase III trial 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 containing PNU-159682 can be conjugated to antibodies via several linking sites and various linkers, including those described herein (US2011 / 0076287, WO2009 / 099741, US2010 / 0034837, WO2010 / 009124).

[0230] The linker of PNU-159682 maleimide acetal-Ab is acid-unstable, while 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 of )-Ab is cleavable with proteases.

[0231] (6) Other drug parts Furthermore, the drug portion includes geldanamycin (Mandler et al., J. Nat. Cancer Inst., vol.92, pp.1573-1581, 2000; Mandler et al., Bioorganic & Med. Chem. Letters, vol.10, pp.1025-1028, 2000; Mandler et al., Bioconjugate Chem., vol.13, pp.786-791, 2002), as well as enzyme-active toxins and their fragments (but not limited to, diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, and Phytolacca americana). This also includes americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitors, curcin, crotin, Sapaonaria officinalis inhibitors, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichoteene. See, for example, WO93 / 21232.

[0232] The drug portion also includes compounds with nucleic acid degradation activity (e.g., ribonucleases or DNA endonucleases).

[0233] In certain embodiments, the immunoconjugate may contain highly radioactive atoms. Various radioisotopes are available for the production of radioconjugated antibodies. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212Examples include radioactive isotopes of Lu. In some embodiments, when an immunoconjugate is used for detection, it may be a radioactive atom for scintigraphy studies, such as Tc 99 Or I 123 or spin labeling for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging or MRI), which may include, for example, 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 with various metal chelating agents and conjugated to antibodies for, for example, PET imaging (WO2011 / 056983).

[0234] Radiolabeling or other labeling may be incorporated into the immunoconjugate by known methods. For example, peptides may be biosynthesized or chemosynthesized using suitable amino acid precursors containing, for example, one or more fluorine-19 atoms instead of one or more hydrogen atoms. In some embodiments, Tc 99 , I 123 Re 186 Re 188 , and In 111 Labels such as can be bound via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be bound via lysine residues in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0235] In certain embodiments, the immunoconjugate may include an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., peptidyl chemotherapeutic agents, see WO81 / 01145) into an active drug such as an anticancer agent. Such immunoconjugates are useful in some embodiments in antibody-dependent enzyme-mediated prodrug therapy ("adept"). Enzymes that can be conjugated to an antibody include, but are not limited to, alkaline phosphatases useful for converting phosphate-containing prodrugs into free drugs, arylsulfatases useful for converting sulfate-containing prodrugs into free drugs, cytosine deaminases useful for converting harmless 5-fluorocytosine into the anticancer drug 5-fluorouracil, serratia proteases, pyrolysis, subtilisin, carboxypeptidases and cathepsins (such as cathepsin B and L), and proteases useful for converting peptide-containing prodrugs into free drugs. Examples include D-alanyl carboxypeptidase, β-galactosidase, and neuraminidase, which are useful for converting prodrugs containing acid substituents; carbohydrate-cleaving enzymes, useful for converting glycosylated prodrugs into free drugs; β-lactamases, useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are useful for converting drugs derivatized with amine nitrogen having a phenoxyacetyl group or a phenylacetyl group into free drugs. In some embodiments, the enzymes may be covalently bound to an antibody by recombinant DNA technology, which is well known in the art. See, for example, Neuberger et al., Nature, vol.312, pp.604-608, 1984.

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

[0237] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, if the binding is to a cysteinethiol, as in some exemplary embodiments described above, the antibody may have only one or more cysteinethiol groups, or only one or more sufficiently reactive thiol groups to which the linker can bind. In certain embodiments, higher drug loadings, e.g., p > 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the average drug loading of an ADC is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In fact, for certain ADCs, the optimal ratio of drug portion per antibody may be less than 8 and may be about 2 to about 5 (U.S. Patent No. 7,498,298).

[0238] In certain embodiments, during the conjugation reaction, less than the theoretical maximum value of the drug moiety is conjugated to the antibody. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as considered below. Generally, antibodies do not contain many free and reactive cysteinethiol groups that can be linked to the drug moiety. In fact, most cysteinethiol residues in antibodies exist as disulfide crosslinks. In certain embodiments, the antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteinethiol groups under partially or entirely reducing conditions. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophiles such as lysine or cysteine.

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

[0240] It should be understood that when two or more nucleophiles react with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADCs having a distribution in which one or more drug moieties are bound 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 to both the antibody and the drug. Individual ADCs in the mixture can be identified by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (see, e.g., McDonagh et al., Prot.Engr.Design & Selection, vol.19, pp.299-307, 2006; Hamblet et al., Clin.Cancer Res., vol.10, pp.7063-7070, 2004). In certain embodiments, a homogeneous ADC with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0241] iv) Specific methods for preparing immunoconjugates Immunoconjugates, which are ADCs of formula I, can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting the nucleophile of the antibody with a divalent linker reagent to form Ab-L via covalent bonding, followed by reaction with the drug moiety D; and (2) reacting the nucleophile of the drug moiety with a divalent linker reagent to form DL via covalent bonding, followed by reaction with the nucleophile of the antibody. An exemplary method for preparing an ADC of formula I via the latter route is described in U.S. Patent No. 7,498,298.

[0242] Nucleophiles on antibodies 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 to which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophiles on the linker moiety and linker reagent, including: (i) active esters such as NHS esters, HOBt esters, halogates, and acid halides, (ii) alkyl and benzyl halides such as haloacetamides, and (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​crosslinks. Antibodies can be made reactive for conjugation with linker reagents by treating them with reducing agents such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the antibody is completely or partially reduced. Therefore, each cysteine ​​crosslink theoretically forms two reactive thiol nucleophiles. Additional nucleophiles can be introduced into the antibody, for example, through modification of the lysine residue by reacting the lysine residue with 2-iminothiolane (Trout's reagent), thereby converting the amine to a thiol. Alternatively, reactive thiol groups may be introduced into the antibody by introducing one, two, three, four, or more cysteine ​​residues (for example, by preparing a variant antibody containing one or more non-natural cysteine ​​amino acid residues).

[0243] The antibody-drug conjugates of the present invention can also be generated by a reaction between an electrophile on an antibody or antibody fragment, such as an aldehyde group or a ketone carbonyl group, and a nucleophile on a linker reagent or drug. Useful nucleophiles on linker reagents include, but are not limited to, hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides. In one embodiment, the antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on the linker reagent or drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, with a periodic acid oxidizing reagent to form an aldehyde or ketone group that can react with an amine group on the linker reagent or drug moiety. The resulting imine Schiff base can form a stable bond, or can be reduced, for example, with a boron hydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate may result in a carbonyl (aldehyde and ketone) group in the antibody that can react with a suitable group on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue may react with sodium metaperiodate, resulting in the production of an aldehyde instead of a first amino acid (Geoghegan & Stroh, Bioconjugate Chem., vol.3, pp.138-146, 1992, U.S. Patent No. 5,362,852). Such an aldehyde may react with the drug moiety or a linker nucleophile.

[0244] Examples of nucleophiles on the drug moiety include, but are not limited to, amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazide groups that can react to form covalent bonds with electrophiles on the linker moiety, as well as the following linker reagents: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups.

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

[0246] In yet another embodiment, an antibody or antibody fragment 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 the removal of unbound conjugates from circulation using a scavenging agent, and then the administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

[0247] C. Methods and compositions for diagnosis and detection In certain embodiments, either the anti-CTLA4 antibody or antibody fragment provided herein may be used to detect the presence of CTLA4 in a biological sample. As used herein, the term “detect” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues such as those of the breast, pancreas, esophagus, lungs, and / or brain.

[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 preferred embodiments, the antibody or antibody fragment of the present invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule, radioactive molecule, or any other label known in the art, as described above. For example, the antibody or antibody fragment of the present invention may 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 Examples of radioactive atoms used in scintigraphy studies include Re. Furthermore, the antibodies or antibody fragments of the present invention may also be labeled with spin labeling for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound.

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

[0251] The antibodies or antibody fragments of the present invention may be useful in diagnosing non-cancerous diseases 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 encompass a variety of sample types obtained from subjects that can be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other fluid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their offspring. For example, biological samples include cells obtained from tissue samples collected from individuals suspected of having 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 encompass clinical samples, cells in culture, cell supernatant, cell lysates, serum, plasma, biological fluids, and tissue samples.

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

[0253] To monitor cancer progression, the method according to the present invention may be repeated at different time points 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 relates to a method for diagnosing diseases associated with the expression or overexpression of CTLA4, or a decrease or increase in the soluble form of CTLA4. Examples of such diseases may include human immunodeficiencies, 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 diagnostic or detection method. In a further embodiment, a method for detecting the presence of CTLA4 in a biological sample is provided. In a further embodiment, a method for quantifying the amount of CTLA4 in a biological sample is provided. In a particular embodiment, the method includes contacting a biological sample with the 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 includes administering the anti-CTLA4 antibody or antibody fragment to the subject.

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

[0257] D. Pharmaceutical preparations Anti-CTLA4 antibodies or antibody fragments possess cell-killing activity. This cell-killing activity extends to several different cell lines. Furthermore, once conjugated with cytotoxic agents, these antibodies or antibody fragments can reduce tumor size and exhibit reduced toxicity. Therefore, anti-CTLA4 antibodies, their fragments, or immunoconjugates may be useful in the treatment of proliferative disorders associated with CTLA4 expression. Antibodies, fragments, or immunoconjugates can be used alone or in combination with any suitable agent or other conventional treatment.

[0258] Anti-CTLA4 antibodies or antibody fragments can be used to treat diseases associated with CTLA4 expression, overexpression, or activation. There are no particular limitations on the types of cancer or tissues that can be treated other than those related to 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, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, sarcoma, hematological cancer (leukemia), astrocytoma, and various types of head and neck cancers. 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 the innate immune response and can therefore be used in the treatment of human immunological diseases such as sepsis. Furthermore, the anti-CTLA4 antibodies or antibody fragments of the present invention can be used as adjuvants for immunization, such as vaccines, and can also be used as anti-infective agents against bacteria, viruses, and parasites, for example.

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

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

[0262] For administration, anti-CTLA4 antibodies, antibody fragments, or immunoconjugates may be formulated as pharmaceutical compositions. Pharmaceutical compositions containing anti-CTLA4 antibodies, antibody fragments, or immunoconjugates can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0263] A pharmaceutically acceptable carrier is a substance that is tolerable by the recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further contain 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 a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc. These considerations can be taken into account by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical compositions 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 pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline (such as monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, or mixtures of such salts), or dry (especially lyophilized) compositions, which enable the formation of an injectable solution, for example, when sterile water or saline is added.

[0266] In some embodiments, isotonic agents, sometimes also 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 reduce the potential for intermolecular and intramolecular interactions by interacting with the charged groups of amino acid side chains. Isotonic agents may be present in any amount of 0.1 to 25% by weight, preferably 1 to 5% by weight, of the pharmaceutical composition. Preferred isotonic agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0267] Further excipients include agents that can function as one or more of the following: (1) volume extenders, (2) dissolution accelerators, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above), amino acids (e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine), organic sugars or sugar alcohols (e.g., sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinitose, myonititol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene glycol), Examples 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] Nonionic surfactants or detergents (also known as "wetting agents") can be used to help solubilize the therapeutic agent and protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface loading without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

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

[0270] The dose used for administration is adapted as a function of various parameters, particularly 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 antibody or antibody fragment can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0271] Suitable dosage forms for injection include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or propylene glycol aqueous solutions, and sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0272] Solutions of active compounds, either as free bases or pharmacologically acceptable salts, can be prepared in water, preferably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0273] Anti-CTLA4 antibodies or antibody fragments can be formulated into neutral or salt-formulated compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid). Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine).

[0274] The carrier may also be a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained by maintaining the required particle size, for example, by using a coating such as lecithin, and by using a surfactant. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injectable composition can be provided by using absorption-delaying agents in the composition, for example, aluminum monostearate and gelatin.

[0275] Sterile injection solutions are prepared by incorporating the required amount of the active compound into a suitable solvent containing, if necessary, one or more components other than those listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilizing active ingredients into a sterile vehicle, comprising a basic dispersion medium and other components required from those listed above. In the case of sterilizing powders for preparing sterilizing injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, from which powders of the active ingredient and any additional desired components are obtained from a pre-filtered sterilized solution.

[0276] Furthermore, the preparation of larger or higher-concentration solutions for direct injection is also being considered. By using dimethyl sulfoxide (DMSO) as the solvent, it is anticipated that very rapid penetration will be achieved, allowing for the delivery of high concentrations of the active agent to small tumor areas.

[0277] During formulation, the solution is administered in a form compatible with the drug formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solutions described above, but drug-releasing capsules can also be used.

[0278] For parenteral administration in aqueous solutions, the solution may be appropriately buffered as needed, and the liquid diluent may first be isotonicized with sufficient saline or glucose. These particular aqueous solutions are especially 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 may 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, e.g., “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Several variations in dosage will inevitably arise depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0279] Antibodies or antibody fragments may be formulated within a therapeutic mixture to deliver approximately 0.0001 to 10.0 mg, or approximately 0.001 to 5 mg, or approximately 0.001 to 1 mg, or approximately 0.001 to 0.1 mg, or approximately 0.1 to 1.0 mg, or even approximately 10 mg per single dose. Multiple doses may also be administered at selected time intervals.

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

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

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

[0283] Liposomes are dispersed in an aqueous medium and are composed of phospholipids that spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs creates small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an 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 anti-CTLA4 antibodies or antibody fragments as described herein are prepared by mixing such antibodies or antibody fragments of desired purity, in the form of lyophilized formulations or aqueous solutions, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and are not limited to, but include buffers (such as phosphoric acid, citrate, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzylammonium chloride), hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), and low molecular weight polypeptides (approximately 1 Examples include less than 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 dextrin), 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 nonionic surfactants (such as polyethylene glycol (PEG)).

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

[0286] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody preparations are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains histidine-acetate buffer.

[0287] The formulations described herein may also contain two or more active ingredients for the specific indication being treated, as needed. Preferably, ingredients having complementary activity that does 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 preferably 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 distinct molecules, namely the anti-CTLA4 antibody, antibody fragment, or immunoconjugate of the present invention and the other antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule, having binding affinity to both CTLA4 and the other antigen, and thus forming a multispecific (e.g., bispecific) antibody.

[0289] The active ingredient may be encapsulated in microcapsules 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 colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0290] Sustained-release preparations can be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody or antibody fragment, the matrix of which may be in the form of a molded article, such as a film or microcapsules.

[0291] Preparations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0292] E. Treatment methods and compositions Any anti-CTLA4 antibody or antibody fragment provided herein can be used in a therapeutic method. In one embodiment, an anti-CTLA4 antibody or antibody fragment is provided for use as a drug. In a further embodiment, an anti-CTLA4 antibody or antibody fragment is provided for use in the treatment of cancer (e.g., breast cancer, non-small cell lung 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 therapeutic method. In a particular embodiment, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having cancer, comprising administering an effective amount of the anti-CTLA4 antibody or antibody fragment to the individual. In certain embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in a method of treating an individual having an immunodeficiency (e.g., autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering an effective amount of the anti-CTLA4 antibody or antibody fragment to the individual. In such one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one described below) to the individual. In further embodiments, the present invention provides an anti-CTLA4 antibody or antibody fragment for use in inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., tumor-associated macrophage-derived), tumor vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or tumor stromal function.

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

[0294] In further embodiments, the present invention provides the use of anti-CTLA4 antibodies or antibody fragments in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of cancer (in some embodiments, breast cancer, non-small cell lung 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 further embodiments, the pharmaceutical is for use in a method of treating cancer, comprising administering an effective amount of the pharmaceutical to an individual having cancer. In further embodiments, the pharmaceutical is for use in a method of treating immunodeficiencies (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 anti-CTLA4 antibodies or antibody fragments to an individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one of those described below) to an individual. 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 vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), 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 vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or tumor stromal function in an individual, comprising administering an effective amount of the agent to an individual to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine fragments (e.g., from tumor-associated macrophages), inhibit the development of tumor vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or inhibit tumor stromal function. The "individual" in any of the above embodiments may be a human being.

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

[0296] In further embodiments, the present invention provides methods for treating immunodeficiencies (e.g., autoimmune disorders), cardiovascular disorders (e.g., atherosclerosis, hypertension, thrombosis), infectious diseases (e.g., Ebola virus, Marburg virus), or diabetes. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one described below) to an individual. The “individual” in any of the above embodiments may be a human.

[0297] In further embodiments, the present invention provides methods for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., tumor-associated macrophages), tumor vascular structures (e.g., intratumor vascular structures or tumor-associated vascular structures), and / or tumor stromal function in an organism. In one embodiment, the method involves administering an effective amount of anti-CTLA4 antibody or antibody fragment to an organism to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce fragments of inflammatory cytokines (e.g., tumor-associated macrophages), inhibit the development of tumor vascular structures (e.g., intratumor vascular structures or tumor-associated vascular structures), and / or tumor stromal function. In one embodiment, “organism” is a human.

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

[0299] In each of the treatments described above and in each of the treatments, the antibody or antibody fragment of the present invention may be used alone, as an immune conjugate, or in combination with other agents during treatment. For example, the antibody of the present invention may be administered concurrently 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 cell division inhibitor. 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 therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) as well as individual administrations, in which case the administration of antibodies or antibody fragments may occur before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants. Antibodies or antibody fragments may also be used in combination with radiotherapy.

[0301] Anti-CTLA4 antibodies or antibody fragments may be formulated, administered, and given in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the physician. Antibodies or antibody fragments may be formulated, optional but not always necessary, with one or more drugs currently used to prevent or treat the disorder in question. The effective dose of such other drugs depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors considered above. These are generally used in the same doses and routes of administration described herein, or at approximately 1–99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

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

[0303] Specific doses of the anti-CTLA4 antibody or antibody fragment of the present invention, which may be administered for the prevention or treatment of the target disease, may be approximately 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 patient weight. In certain embodiments, the dose may range from 0.3 to 2.4, 2.4 to 4.2, 4.2 to 6.0, 6.0 to 7.8, 7.8 to 10.2, 10.2 to 12, 12 to 14, 14 to 16, 16 to 18, or 18 to 20 mg of antibody or antibody fragment / kg patient weight. When administered in the form of a bispecific antibody in combination with another immune checkpoint inhibitor or another antibody or antibody fragment, or as an immune conjugate, the dose of the antibody or antibody fragment remains the same. Furthermore, polypeptides possessing anti-CTLA4 activity are administered in the same amount as the antibody or antibody fragment.

[0304] A single dose of the pharmaceutical formulation of the present invention may contain an amount of the anti-CTLA4 antibody or antibody fragment of the present invention from about 45 μg of antibody or antibody fragment, about 45 μg of antibody or antibody fragment from about 13,600 mg, or about 45 μg of antibody or antibody fragment from about 5,440 mg. In some embodiments, a single dose of the pharmaceutical formulation of the present invention may contain an amount of 135 mg to 1,387 mg, or an amount such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg of the anti-CTLA4 antibody or antibody fragment of the present invention. 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 is in the range of 135-235, 235-335, 335-435, 435-535, 535-635, 635-735, 735-835, 835-935, 935-1035, 1035-1135, 1135-1235, and 1235-1387 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 immune conjugate, 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 the same amount as the antibody or antibody fragment in a single dose of the pharmaceutical formulation.

[0305] In one embodiment, 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] Other immune checkpoint inhibitor molecules may be antibodies or antibody fragments against immune checkpoints other than CTLA4. The combination may include the anti-CTLA4 antibody or antibody fragment disclosed herein, as well as other immune checkpoint inhibitor molecules administered as separate molecules or as bispecific antibodies. Such bispecific antibodies have binding activity to CTLA4 and a second binding activity to another immune checkpoint.

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

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

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

[0310] Enhancing the host's immune function to combat tumors is a subject of growing interest. Conventional methods include (i) enhancement of APCs, e.g., (a) injecting the tumor with DNA encoding an alloantigen of exogenous MHC, or (b) transfection of biopsy tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), and (iii) adoptive cell immunotherapy or treatment with activated tumor-specific T cells. Adoptive cell immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, e.g., through stimulation by IL-2, the tumor, or both. Furthermore, the isolated, dysfunctional T cells can also be activated by in vitro application of the anti-PD-L1 antibody of the present invention. The thus activated T cells may then be re-administered to the host. One or more of these methods can be used in combination with the administration of the antibody, antibody fragment, or immune conjugate of the present 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 radiotherapy (EBRT) or via internal close-range radiotherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or drugs that specifically affect dysregulated 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., if the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking of blood vessel formation and growth; and (vii) palliative care, or treatment aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant, can enable more aggressive treatment regimens.

[0312] In cancer treatment, any of the aforementioned conventional therapies for cancer immunotherapy may be administered before, after, or concurrently with the administration of anti-CTLA4 antibodies or antibody fragments. In addition, anti-CTLA4 antibodies or antibody fragments may be administered before, after, or concurrently with conventional cancer therapies such as the administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or chemotherapy agents.

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

[0314] It should be understood that any of the above products may contain the immunoconjugate of the present invention in place of, or in addition to, an anti-CTLA4 antibody or antibody fragment.

[0315] Finally, the present invention also provides a kit comprising at least one antibody or antibody fragment of the present invention. A kit comprising the polypeptide, antibody or antibody fragment, or antibody-drug conjugate of the present invention is useful for detecting (increasing or decreasing) CTLA4 expression, or for therapeutic or diagnostic assays. 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 comprising an antibody for in vitro detection and quantification of CTLA4 (e.g., ELISA or Western blotting) can be provided. Such antibodies useful for detection may be provided with labeling, such as fluorescent or radiolabeling.

[0316] The kit further includes 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 kit further includes instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of CTLA4 in the sample. In some embodiments, the kit includes one or more antibodies or antibody fragments. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors or enzyme activators. In yet another embodiment, the kit further includes one or more chromatographic compounds. In yet another embodiment, the kit further includes one or more compounds used to prepare a sample for a spectroscopic assay. In a further embodiment, the kit further includes 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 illustrate, but are not limited to, the soft gelatin capsules of the present disclosure. Various other suitable modifications and adaptations of conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the scope of the present disclosure. [Examples]

[0318] Example 1: Conditionally active biological (CAB) antibody against CTLA4 In this embodiment, antibodies against CTLA4 were produced (Table 2). [Table 2]

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

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

[0321] Table 3-4 shows the EC2 binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 at pH 6.0 and pH 7.4. 50The values ​​are shown, and the binding curves for representative experiments 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 compared to ipilimumab and ipilimumab analogs, but showed significantly reduced binding activity at pH 7.4. [Table 3] [Table 4]

[0322] In addition, the binding activity of BA-087-05-19 and BA-087-08-32 to the immobilized recombinant cynomolgus monkey CTLA4 extracellular domain was also determined by ELISA. Table 5 shows the EC2 binding activity of BA-087-05-19 and BA-087-08-32 to cynomolgus monkey CTLA4 at pH 6.0. 50 Figures 4A and 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] EC of the binding activity of BA-087-05-19 and BA-087-08-32 at pH 6.0 in the tumor microenvironment as measured by ELISA. 50 The EC levels for human CTLA4 were found to be 8.18 ng / mL and 9.78 ng / mL, respectively, and the EC levels for ipiliumumab and ipiliumumab analogs were determined. 50The results were similar. BA-087-05-19 had similar binding activity to both human CTLA4 and cynomolgus monkey CTLA4 at pH 6.0, while BA-087-08-32 showed decreased binding activity to cynomolgus monkey CTLA4 at pH 6.0 compared to its binding activity to human CTLA4. The decrease in binding activity of BA-087-08-32 to cynomolgus monkey CTLA4 at pH 6.0 observed in ELISA appears to be specific to the ELISA assay, as the same decrease was not observed using either SPR or FACS. The binding activity of BA-087-05-19 and BA-087-08-32 to human CTLA4 or cynomolgus monkey CTLA4 at normal physiological pH 7.4, as measured by ELISA, was significantly lower than the binding activity at pH 6.0.

[0324] Example 3: pH-dependent binding activity of anti-CTLA4 antibody The binding activity of antibodies to CTLA4 was tested using an ELISA assay in the pH range of 5.0–7.4. Recombinant human CTLA4 extracellular domains were immobilized in wells in pH buffer (pH 5.0–pH 7.4) to mimic the pH of the tumor microenvironment (pH 5.5–pH 6.7) and normal physiological pH (pH 7.4), and 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 domains was measured. The amounts of bound antibodies BA-087-05-19 and BA-087-08-32 were quantified using anti-human IgG antibodies conjugated with horseradish peroxidase (HRP), and then reacted with a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to produce colored products. 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, and 90% binding activity was present 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, and 90% binding activity was present at pH 6.2.

[0326] The average OD values ​​at different pH levels (from two iterations) were plotted against the pH of the buffer using Softmax Pro software (Molecular Devices). Curve fitting was performed using the software's built-in 4-parameter model. The inflection point of the pH curve (= 50% binding activity at pH 6.0) is equal to parameter C in the fitting equation. 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 values ​​for BA-087-05-19 and BA-087-08-32 at 50% and 90% activity were calculated using the pH values ​​obtained in Experiments 1-4. Experiments 1-4 used BA-087-05-19 (lot number #6972) and BA-087-08-32 (lot number #6978). Experiment 5 used BA-087-05-19 (lot number #6901) and BA-087-08-32 (lot number #6902) from a different lot. The pH values ​​for BA-087-05-19 and BA-087-08-32 at 50% and 90% activity determined from the data in Experiment 5 were similar to the average pH values ​​calculated using the pH values ​​from 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 the positive control ipilimumab and ipilimumab analogs, to recombinant human CTLA4 in various pH buffers. The pH-dependent binding inflection points for BA-087-05-19 and BA-087-08-32 were calculated to be pH 6.97 and pH 6.43, respectively. 90% 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 the normal physiological pH of 7.4 (Figure 5).

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

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

[0331] Furthermore, BA-087-08-32 showed sub-nanomolecal binding activity at pH 6.0. Binding activity decreased approximately 100-fold from pH 6.0 to pH 7.4 (K D [pH6.0] = 0.45 nM, K D [pH7.4] = 45 nM). In addition to the lower binding activity at pH7.4, the SPR signal at pH7.4 reaches only about 10% of the signal level at pH6.0, indicating that only a very small number of BA-087-08-32 molecules are capable of binding to human CTLA4 at pH7.4. See Table 8. [Table 8]

[0332] When the commercially available anti-CTLA4 antibody ipilimumab (Yervoy®) was used as a control under the same conditions, the binding activity was found to be very similar at pH 6.0 and pH 7.4 (K D [pH6.0] = 1.39 nM, K D [pH7.4] = 1.37 nM). Therefore, the binding activity of ipilimumab was pH-independent. See Table 9. In addition, the obtained SPR signals were 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 for human CTLA4 using the same conditions as described above. All three antibodies exhibited a fast off rate, and the SPR signal reached equilibrium at all antibody concentrations tested. D This was calculated by plotting the maximum SPR signal at each antibody concentration against the antibody concentration. The experiment was performed three times at each pH.

[0334] Antibody BA-087-05-19 has a K content of 1.96 nM at pH 6.0. D It binds to CTLA4 in cynomolgus monkeys. At pH 7.4, the calculated value is K D It shows >100 nM. Antibody BA-087-08-32 has a K content of 5.95 nM at pH 6.0. D It binds to CTLA4 in cynomolgus monkeys. At pH 7.4, the obtained SPR signal is K D It is too low to calculate. The antibody ipilimumab has a K content of 6.58 nM at pH 6.0. D、 And at pH 7.4, the K content is 6.80 nM. D It then binds to CTLA4 in cynomolgus monkeys.

[0335] Example 5: FACS analysis of anti-CTLA4 antibody The binding activity of antibodies BA-087-05-19 and BA-087-08-32 to human CTLA4 and cynomolgus monkey CTLA4 expressed on the cell surface of CHO cells was measured by FACS in buffers at pH 6.0 or pH 7.4. Serially diluted BA-087-05-19, BA-087-08-32, ipilimumab, and ipilimumab analogs were added to CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4. The amount of antibody bound to the cells was quantified using anti-human IgG antibody conjugated to a fluorophore. EC values ​​for cell binding at pH 6.0 and 7.4 were also measured. 50 The values ​​were calculated using a nonlinear fitted (variable slope, 4-parameter) model incorporated into GraphPad Prism software (version 7.03). The expression levels of human CTLA4 or cynomolgus monkey CTLA4 on the surface of CHO cells were determined using the BD QuantiBRITE® PE kit.

[0336] For each antibody, at least two independent duplicate FACS experiments were performed using each cell line. Figures 6A-6B show the binding activity of the antibody to human CTLA4 on CHO cells (CHO-huCTLA4) at pH 6.0 and 7.4. Figures 7A-7B show the binding activity of the antibody to cynomolgus monkey CTLA4 on CHO cells (CHO-cynoCTLA4) at pH 6.0 and 7.4. These figures plot the binding activity at different concentrations of the antibody.

[0337] The binding activity of BA-087-05-19 and BA-087-08-32 at pH 6.0, as measured by FAC, was 350.1 and 243.4 ng / mL for human CTLA4, and 316.2 and 402.6 ng / mL for cynomolgus monkey CTLA4, respectively, with mean EC2 levels. 50 It was found that it possesses the following properties. The binding activity of ipilimumab and ipilimumab analogs at pH 6.0, as measured by FAC, was 341.1 ng / mL and 325.4 ng / mL for human CTLA4, and 337.5 ng / mL and 319.6 ng / mL for cynomolgus monkey CTLA4, respectively, with an average EC2 of 3. 50 It was found that 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 monkey CTLA4 at pH 6.0 with similar affinity to ipilimumab and ipilimumab analogs. However, BA-087-05-19 and BA-087-08-32 exhibit significantly weaker binding activity to human and cynomolgus monkey CTLA4 at pH 7.4 compared to ipilimumab and ipilimumab analogs at pH 7.4, respectively. No binding activity was detected in CHO cells that did not express CTLA4.

[0339] Finally, at pH 7.4, the saturation of antibodies on CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4 was also measured by FACS (Figures 8A-8B). At pH 7.4, antibodies BA-087-05-19 and BA-087-08-32 bound to CHO cells less than 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 and pH 7.4. Binding activity was measured using both ELISA and FACS analysis. In ELISA analysis, serially diluted samples of BA-087-05-19 and BA-087-08-32 were added to wells of their respective buffers, pre-coated with human CTLA4. The amount of bound antibody was quantified using HRP-conjugated anti-human IgG antibody. 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 The values ​​(ng / mL) were determined by the absorbance at 450 nm relative to antibody concentration using a 4-parameter dose-response curve of the Prism variable gradient, which was calculated using a nonlinear fitted (variable gradient, 4 parameters) model incorporated into GraphPad Prism software (version 7.03). Tables 10-11 show the EC of binding to human CTLA4 measured by ELISA in different buffers. 50 The values ​​are shown. The buffers tested included His buffer (His), Tris buffer (Tris), glutamine buffer (Glu), and no buffer. [Table 10] [Table 11]

[0341] In FACS analysis, serially diluted BA-087-05-19 and BA-087-08-32 samples were added to cells expressing human CTLA4. The amount of bound antibody was quantified using anti-human IgG antibody conjugated to a fluorophore. The MFI in each reaction was proportional to the amount of bound antibody. The binding activity measured by FACS is shown in Figures 10A-10F. EC for binding to human CTLA4 on cells. 50 The values ​​(ng / mL) were determined by the MFI of the singlet population against antibody concentration using a 4-parameter dose-response curve with a variable gradient in Prism, which was calculated using a nonlinear fitted (variable gradient, 4-parameter) model incorporated into GraphPad Prism software (version 7.03). EC for binding to human CTLA4 on CHO cells. 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 determined the potential immunogenicity of BA-087-05-19 using the EpiVax in silico immunogenicity screening toolkit. The software was accessed via ISPRI, a web-based interactive screening and protein redesign interface. Using the BA-087-05-19 variable domain as input, the software was used to assess potential immunogenicity on a normalized scale and predict potential ADA response.

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

[0344] Example 8: Functional assay of IL-2 secretion enhanced by anti-CTLA4 antibody In this example, the functional activity of antibodies BA-087-05-19 and BA-087-08-32 in inducing IL-2 secretion by human lymphocytes stimulated by Staphylococcus enterotoxin B (SEB) was determined. Serially diluted BA-087-05-19, BA-087-08-32, ipilimumab, and ipilimumab analogs were added to normal, healthy donor-derived human peripheral blood mononuclear cells (PBMCs) stimulated with SEB. The ability of the antibodies to enhance IL-2 secretion in SEB-stimulated human PBMCs was quantified using an IL-2 ELISA kit.

[0345] A total of three independent experiments were conducted. 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 to levels observed with the addition of isotype controls at levels observed with ipilimumab and ipilimumab analogs at pH 6.2. On the other hand, as shown in Figure 11B, IL-2 production did not increase with the addition of BA-087-05-19 and BA-087-08-32 at pH 7.4.

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

[0347] Example 9: Promega® CTLA4 Blockade Assay for Anti-CTLA4 Antibody 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 using the in vitro Promega® CTLA4 blockade assay. Serially diluted BA-087-05-19, BA-087-08-32, ipilimumab, and ipilimumab analogs were added to Jurkat effector cells, followed by the addition of aAPC / Raji cells according to the vendor's protocol. Blockade of the interaction between CTLA4 and its ligand resulted in activation of a modified IL-2 pathway 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 could block the interaction between CTLA4 and its ligand (CD80 / CD87) at pH 6.0 at a similar level to that observed with ipilimumab and ipilimumab analogs. In contrast, as shown in Figure 12B, BA-087-05-19 and BA-087-08-32 were less effective at blocking the interaction between CTLA4 and its ligand 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 significantly lower than that of ipilimumab and ipilimumab when blocked at pH 7.4.

[0349] Example 10: FACS assay for ligand blockade using anti-CTLA4 antibody The activity of BA-087-05-19 and BA-087-08-32 in inhibiting the interaction of human CTLA4 with its ligands hB7-1 (hCD80) and hB7-2 (hCD86) was assayed by FACS, and competitive binding of BA-087-05-19 and BA-087-08-32 to human CTLA4-expressing CHO cells at fixed concentrations in the presence of different concentrations of hB7-1 and hB7-2 was evaluated. The amount of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4 cells was quantified using anti-human IgG antibodies conjugated to fluorophores. As shown in Figures 13A to 13B, the mean fluorescence intensity (MFI) in each reaction was proportional to the amount of BA-087-05-19 and BA-087-08-32 bound to CHO-huCTLA4.

[0350] In addition, competitive binding of serially diluted BA-087-05-19 and BA-087-08-32 to human CTLA4-expressing CHO cells was determined using FACS analysis at fixed concentrations of hB7-1 and hB7-2. The amounts of hB7-1 and hB7-2 bound to CHO-huCTLA4 cells were quantified using anti-His antibody and 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 and its ligands with hB7-1 and hB7-2 at levels similar to those achieved by ipilimumab and ipilimumab analogs (Figures 14A-14B).

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

[0352] Method used in the examples The ELISA assay was performed using the following protocol: 1) Coat the ELISA plate with recombinant CTLA4 antigen at a concentration of 0.5 μg / mL (experiments 06_20_17 and 06_28_17) or 1 μg / mL (experiments 07_06_17 and 07_11_17) in 100 μL of carbonic acid-bicarbonate coating buffer. 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently 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 each well according to the sample map, and then aspirate the contents completely. 5) Add 200 μL of various pH incubation buffers to the wells according to the sample map. Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 7) Serially dilute the test substance in various pH incubation buffers to 250 ng / mL, 100 ng / mL, or 25 ng / mL. 8) Add 100 μL / well of diluted test material to the plate according to the sample map. 9) Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and gently 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 washing buffers into each well according to the sample map and completely aspirating the contents. 12) Dilute the HRP secondary antibody to 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 it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and gently 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 washing buffers into each well according to the sample map and completely aspirating the contents. 17) Dispense 50 μL of TMB substrate solution into each well of the plate. Incubate at room temperature for 3 minutes. 18) Add 50 μL of 1N HCl per well to all wells of the plate. Read the plate at 450 nm using a Molecular Device SpectraMax 190 microplate reader. 19) Measure the raw data at OD450nm. 20) Using Softmax Pro software (Molecular Devices), the average OD values ​​(from two replicates) at different pH levels were plotted against the pH of the buffer. Curve fitting was performed using the 4-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. 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] The following protocol was used to perform a surface plasmon resonance (SPR) assay: The SPR2 / 4 instrument, SPR affinity sensor (Amine Flat), and immobilization buffer kit are manufactured by Sirra Sensors. The SPR sensor has four flow cells (FC1-FC4), which can be used 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] The system was fixed according to the vendor's recommended protocol. (1) The activator was prepared by mixing 200 mM EDC and 50 mM NHS (Sierra Sensors) immediately before injection. The amine sensor tip was activated with the mixture at a flow rate of 25 μL / min for 480 seconds. (2) 25 μg / mL of human CTLA4 in 10 mM NaAc (pH 5.0) was injected into FC2 and FC4, respectively, at a flow rate of 25 μL / min for 480 seconds. The tip surface was deactivated by passing 1 M ethanolamine-HCl (Sierra Sensors) through FC1-FC4 at a flow rate of 25 μL / min for 480 seconds. (3) Using the same conditions, but without protein injection, the control surface was activated and deactivated. (4) Before injecting the analyte, the electrophoresis buffer was switched to PBST at the required pH. Before injecting the first analyte, the analyzer was equilibrated with the electrophoresis buffer for 1 hour. (5) All analytes were injected at 25 μL / min at 25°C.

[0355] BA-087-05-19 was diluted in electrophoresis buffer (PBST buffer, pH 6.0 or 7.4) to concentrations of 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 electrophoresis 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 into flow cells 1 and 2 (or 3 and 4) at a flow rate of 25 μL / min for a 240-second association period, followed by a 360-second dissociation period. The analyte electrophoresis was repeated for 6 cycles while increasing the analyte concentration. After each cycle of interaction analysis, the tip surface was regenerated by injecting 6 μL of 10 mM glycine (pH 2.0). Each set was electrophoresed a total of 3 times at the same pH.

[0357] Flow cell 1 (or 3) without immobilized protein was used as a reference surface for reference subtraction. Furthermore, data containing only buffer (0 nM analyte) was subtracted from each electrophoresis. The double-subtracted data was fitted using a 1:1 coupling model with the provided analytical software Analyzer R2 (Sierra Sensors). The molar concentration of the analyte was 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 the surface expression of human CTLA4 or cynomolgus monkey CTLA4 1) Following the vendor's instructions, fill the T-75 flask and culture medium with 3 × 10 6 Seed individual cells. 2) On the day of the FACS analysis, remove and discard the culture medium. 3) Briefly rinse the cell layer with PBS solution. 4) Add 1.5 mL of Detachin solution to each T-75 flask. Wait until the cell layer is dispersed. 5) Add 4.5 mL of culture medium to the corresponding cell line and resuspend the cells by gentle pipetting. 6) Pool the cells and transfer the cell suspension to a 50 mL conical tube. 7) After counting the cells using trypan blue staining, centrifuge at 1500 rpm for 5 minutes at 4°C. 8) Wash the cells once with PBS and divide into 3 x 10 5 Transfer the individual cells into Eppendorf tubes. 9) Add 2 μL of mouse anti-CTLA4 (PE-conjugated mouse IgG1) 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 at room temperature for 10 minutes, then wash the cells once with PBS. 12) Resuspend the cells in 100 μL of PBS and analyze them using a NovoCyte flow cytometer.

[0360] FACS analysis of CHO cells expressing human CTLA4 or cynomolgus monkey CTLA4 using a test antibody. 1) Harvest the cells (as in 3.3, steps 1-7) and wash the cells once with PBS. 2) Add 3 × 10⁶ to FACS buffer at pH 6.0 or pH 7.4. 6 Resuspend the cells in cells / mL. 3) Place 3 × 10⁶ units in 100 μL of pH 6.0 or pH 7.4 FACS buffer in a 96-well U-bottom plate. 5 Aliquot the cells. 4) Centrifuge the cells and discard the buffer solution. 5) The test substance is serially diluted in 3-fold dilutions starting at 10 μg / mL in FACS buffer at pH 6.0 or pH 7.4 (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). 6) Add 100 μL / well of diluted test material to the cells, gently mix the wells, and incubate on ice for 1 hour with shaking (100 rpm). 7) Centrifuge the cells at 1500 rpm at 4°C for 5 minutes. Wash the cells twice with 150 μL of pH 6.0 or pH 7.4 washing buffer. 8) Dilute the goat anti-human IgG AF488 antibody 1:300 with FACS buffer at pH 6.0 or pH 7.4. 9) Add 100 μL of diluted antibody (from the above steps) to the cells and incubate on ice for 45 minutes, protecting from light. 10) Pellet the cells and wash them three times with 150 μL of pH 6.0 or pH 7.4 washing buffer. 11) Fix the cells in 4% PFA diluted in 1XPBS at room temperature for 10 minutes, then wash the cells with 1XPBS. 12) Resuspend the cells in 100 μL of 1XPBS. 13) Analyze cells using a NovoCyte flow cytometer with Ex488nm / Em530nm. Collect at least 20,000 cells.

[0361] FACS data was analyzed using a nonlinear fitted (variable slope, 4-parameter) model built into GraphPad Prism software (version 7.03).

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

[0363] While many features and advantages of the present invention, along with details of its structure and function, have been described above, it should be understood that this disclosure is illustrative only, and detailed modifications may be made, particularly to matters relating to the shape, size, and arrangement of components within the principles of the present invention, to the full extent indicated by the broad general meaning of the terms expressed in the accompanying claims.

[0364] All documents referenced herein are incorporated herein by reference in their entirety or provide disclosures on which particular reliance is placed. The applicant(s) do not intend to dedicate the disclosed embodiments to the public, and any disclosed modifications or alterations are deemed to be part of the invention under the doctrine of equivalents, insofar as they may not be literally included within the claims.

[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. Anti-CTLA4 antibody or antibody fragment selected from antibodies or antibody fragments containing the following: (a) A light chain variable region having the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10; (b) A light chain variable region having the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12; (c) A light chain variable region having the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14; (d) A light chain variable region having the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16; (e) A light chain variable region having the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18; (f) A light chain variable region having the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20; (g) A light chain variable region having the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22; (h) A light chain variable region having the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24; (i) A light chain variable region having the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26; (j) A light chain variable region having the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28; (k) A light chain variable region having the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 30; (l) A light chain variable region having the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 32; (m) A light chain variable region having the amino acid sequence of SEQ ID NO: 33 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 34; (n) A light chain variable region having the amino acid sequence of SEQ ID NO: 35 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 36; and (o) A light chain variable region having the amino acid sequence of SEQ ID NO: 37 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

38.

2. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

10.

3. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

12.

4. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

14.

5. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

16.

6. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

18.

7. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

20.

8. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

22.

9. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

24.

10. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

26.

11. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 27 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

28.

12. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

30.

13. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 31 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

32.

14. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 33 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

34.

15. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 35 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

36.

16. The anti-CTLA4 antibody or antibody fragment according to claim 1, comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 37 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

38.

17. An antibody or antibody fragment according to any one of claims 1 to 16, having a ratio of 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, or at least 20:1 of the binding affinity to the CTLA4 protein under different conditions in the non-tumor microenvironment.

18. The antibody or antibody fragment according to any one of claims 1 to 17, wherein the antibody or antibody fragment is a chimeric antibody, a multispecific antibody, or a humanized antibody.

19. An immunoconjugate comprising an antibody or antibody fragment according to any one of claims 1 to 18.

20. The immune conjugate according to claim 19, wherein the immune conjugate comprises at least one agent selected from chemotherapeutic agents, radioactive atoms, cell division inhibitors, and cytotoxic agents.

21. An immunoconjugate according to any one of claims 19 to 20, wherein an antibody or antibody fragment and at least one drug are covalently bound to a linker molecule, and at least one drug is selected from meitansinoids, auristatin, drastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

22. A pharmaceutical composition comprising an antibody or antibody fragment according to any one of claims 1 to 18, or an immunoconjugate according to any one of claims 19 to 21 and a pharmaceutically acceptable carrier, optionally comprising an isotonic agent.

23. The pharmaceutical composition according to claim 22, which is formulated for single-dose administration and comprises an antibody or antibody fragment according to any one of claims 1 to 18 or an immune conjugate according to any one of claims 19 to 21 in an amount ranging from 135 mg to 235 mg, 235 mg to 335 mg, 335 mg to 435 mg, 435 mg to 535 mg, 535 mg to 635 mg, 635 mg to 735 mg, 735 mg to 835 mg, 835 mg to 935 mg, 935 mg to 1035 mg, 1035 mg to 1135 mg, 1135 mg to 1235 mg, or 1235 mg to 1387 mg.

24. The pharmaceutical composition according to any one of claims 22 to 23, further comprising an immune checkpoint inhibitor molecule different from the antibody or antibody fragment described in any one of claims 1 to 18.

25. The pharmaceutical composition according to claim 24, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.

26. The pharmaceutical composition according to claim 25, wherein the immune checkpoint is selected from LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS, or the immune checkpoint is PD-1 or PD-L1.

27. A pharmaceutical composition according to any one of claims 22 to 26, further comprising an antibody or antibody fragment against an antigen selected from PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins.

28. The pharmaceutical composition according to claim 27, wherein the WNT protein is selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

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