Ipilimumab variant with enhanced specificity due to binding at low pH

Abstract

The present invention provides variant forms of anti-CTLA-4 antibodies, such as ipilimumab, that preferentially bind to CTLA-4 at low pH. Such antibody variants exhibit preferential activity in the tumor microenvironment, an enhanced ratio of anti-tumor response to side effects, and an enhanced therapeutic index.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 835,794, filed April 18, 2019, the contents of which are incorporated herein by reference.

[0002] Sequence Listing The sequence listing submitted electronically hereby is also incorporated herein by reference in its entirety (Filename: 20200401_SEQL_13177WOPCT_GB.txt; Created: April 1, 2020; File Size: 79 KB). [Background technology]

[0003] Background of the Invention The immune system can control tumor progression and mediate tumor regression. This requires the generation and activation of tumor antigen-specific T cells. Multiple T cell costimulatory receptors and T cell negative regulator or co-inhibitory receptors act in concert to control T cell activation, proliferation, and the acquisition or loss of effector function. Among the earliest and best-characterized T cell costimulatory and co-inhibitory molecules are CD28 and CTLA-4. Rudd et al. (2009) Immunol. Rev. 229: 12. CD28 provides a costimulatory signal for T cell receptor engagement by binding to B7-1 and B7-2 ligands on antigen-presenting cells, while CTLA-4 provides a negative signal that downregulates T cell proliferation and function. CTLA-4 also binds to the B7-1 (CD80) and B7-2 (CD86) ligands, but with higher affinity than CD28, and acts as a negative regulator of T cell function through both cell-autonomous (or intrinsic) and cell-non-autonomous (or extrinsic) pathways. CTLA-4 binds to CD8 and CD4 T effector (T) eff Endogenous regulation of T cell function is mediated by inducible surface expression of CTLA-4, resulting in inhibition of T cell activation and T cell proliferation and cytokine production by multivalent binding of B7 ligands on opposing cells. Peggs et al. (2008) Immunol. Rev. 224:141.

[0004] Anti-CTLA-4 antibodies, when cross-linked, inhibit T cell function in vitro. Krummel & Allison (1995) J. Exp. Med. 182:459; Walunas et al. (1994) Immunity 1:405. Regulatory T cells (T) constitutively express CTLA-4. reg ) activates effector T cells (T eff ) function. reg have impaired inhibitory capacity (Wing et al. (2008) Science 322:271), and antibodies that block CTLA-4 interaction with B7 inhibit T reg It can inhibit the function of T cells (Read et al. (2000) J. Exp. Med. 192:295; Quezada et al. (2006) J. Clin. Invest. 116:1935). eff It has also been shown to regulate T cell function through the extrinsic pathway (Corse & Allison (2012) J. Immunol. 189:1123; Wang et al. (2012) J. Immunol. 189:1118). reg and T eff Extrinsic control of T cell function by CTLA-4 occurs through the ability of CTLA-4-positive cells to remove B7 ligands from antigen-presenting cells, thereby limiting their costimulatory capacity. Qureshi et al. (2011) Science 332: 600; Onishi et al. (2008) Proc. Nat'l Acad. Sci. (USA) 105:10113. Antibody blockade of the CTLA-4 / B7 interaction inhibits T cell function by interfering with the negative signal transduced by CTLA-4 binding. eff This endogenous control of T cell activation and proliferation is thought to promote T eff and T regIt can promote the growth of both tumors and immune cells (Krummel & Allison (1995) J. Exp. Med. 182:459; Quezada et al. (2006) J. Clin. Invest. 116:1935). Early studies in animal models showed that antibody blockade of CTLA-4 exacerbated autoimmunity. Perrin et al. (1996) J. Immunol. 157:1333; Hurwitz et al. (1997) J. Neuroimmunol. 73:57. The ability of anti-CTLA-4 to cause regression of established tumors, with extension to tumor immunity, provided a dramatic example of the therapeutic potential of CTLA-4 blockade. Leach et al. (1996) Science 271:1734.

[0005] Ipilimumab, a human anti-human CTLA-4 monoclonal antibody first approved for the treatment of metastatic melanoma, has since been approved for use in other cancers and is in clinical trials for still other cancers. Hoos et al. (2010) Semin. Oncol. 37:533; Hodi et al. (2010) N. Engl. J. Med. 363:711; Pardoll (2012) Nat. Immunol. 13(12): 1129. Ipilimumab has the human IgG1 isotype, which best binds to most human Fc receptors (Bruhns et al. (2009) Blood 113: 3716) and is considered equivalent to mouse IgG2a in terms of the type of Fc receptor activation it binds; however, human IgG1 is not necessarily equivalent to mouse IgG2a in terms of the antibody-dependent cellular cytotoxicity (ADCC) or other effector function it induces, in that it can induce much lower ADCC than IgG2a does in mice. Ipilimumab can mediate ADCC because IgG1 binds to the activating receptor CD16 (FcγRIIIa) expressed by human NK cells and monocytes. The IgG1 isotype ipilimumab was originally isolated directly from a hybridoma and then cloned and expressed in Chinese hamster ovary (CHO) cells. Despite the consideration that isotypes that mediate ADCC and / or CDC would be undesirable for antibodies targeting receptors on T cells sought to upregulate immune responses, antibodies of the IgG1 isotype were retained because they enhanced vaccine responses in cynomolgus macaques and were considered functional.

[0006] Ipilimumab, for example, reduces post-treatment CD4 + and CD8 +It has been shown to increase the number of activated T cells in the blood, as evidenced by a significant increase in HLA-DR expression on the cell surface as well as an increase in absolute lymphocyte counts (Ku et al. (2010) Cancer 116:1767; Attia et al. (2005) J. Clin. Oncol. 23:6043; Maker et al. (2005) J. Immunol. 175:7746; Berman et al. (2009) J. Clin. Oncol. 27(suppl):15s.3020; Hamid et al. (2009) J. Clin. Oncol. 27(suppl):15s.9008), indicating that T cell depletion does not occur peripherally in humans. Ipilimumab exhibits only a moderate level of ADCC of activated T cells using IL-2-activated PBMCs as effector cells (unpublished data); however, the use of T cells as targets is unclear. reg The use of ipilimumab was not tested. reg Although small changes in frequency have been observed (Maker et al. (2005) J. Immunol. 175:7746), ipilimumab intratumoral T reg However, there is evidence that high CD8+ cells in biopsy samples from metastatic melanoma lesions in patients treated with ipilimumab have been shown to be effective against leukemia. + Against T reg A positive correlation between the CD4 ratio and tumor necrosis has been described. Hodi et al. (2008) Proc. Nat'l Acad. Sci. (USA) 105:3005. Furthermore, tumor tissues from ipilimumab-treated bladder cancer patients had higher CD4 than tumors from untreated bladder cancer patients. + Foxp3 + The percentage of T cells was low. Liakou et al. (2008) Proc. Nat'l Acad. Sci. (USA) 105:14987. These results suggest that ipilimumab inhibits T cells at the tumor site. reg This is consistent with the data disclosed herein that mediates a reduction.

[0007] In contrast, tremelimumab is an IgG2 isotype, which does not efficiently bind to Fc receptors, with the exception of the FcγRIIa variant H131 (Bruhns et al. (2009) Blood 113:3716). On the other hand, tremelimumab has the ability to enhance T cell responses by blocking the inhibitory interaction of CTLA-4 and B7, and studies have shown that tremelimumab inhibits T cell proliferation in tumors. reg These findings suggest that tremelimumab may mediate limited depletion of tumor cells, and therefore is predicted to exhibit reduced antitumor activity compared to ipilimumab. Direct comparison of the clinical activity of these two antibodies is difficult due to the different dosing regimens. See, e.g., Ascierto et al. (2011) J. Transl. Med. 9:196. Tremelimumab, like ipilimumab, has demonstrable antitumor activity. Ribas (2010) Semin. Oncol. 37(5):450. Interestingly, studies of the mechanism of action of tremelimumab have shown, in a limited number of immunohistochemically analyzed samples, that treatment results in an increase in tumor-infiltrating CD8 T cells, while there is no significant increase in Foxp3 T cells in tumors after treatment. + The number of cells remains unchanged. Comin-Anduix et al. (2008) J. Transl. Med. 6:22; Huang et al. (2011) Clin. Cancer Res. 17:4101. Alternatively, T reg Inhibition of function can be achieved by blocking the CTLA-4 / B7 interaction.

[0008] Human antibodies against human CTLA-4, ipilimumab and tremelimumab, were selected for their inhibition of CTLA-4-B7 interaction (Keler et al. (2003) J. Immunol. 171:6251; Ribas et al. (2007) Oncologist 12:873) and have been tested in various clinical trials for a number of malignancies. Hoos et al. (2010) Semin. Oncol. 37:533; Ascierto et al. (2011) J. Transl. Med. 9:196. While tumor regression and disease stabilization are often observed, treatment with these antibodies is accompanied by adverse events of inflammatory infiltrates that can affect various organ systems. In 2011, ipilimumab, which contains an IgG1 constant region, was approved in the US and EU for the treatment of unresectable or metastatic melanoma based on improved overall survival in a Phase III trial of previously treated patients with advanced melanoma. Hodi et al. (2010) N. Engl. J. Med. 363:711. Summary of the Invention [Problem to be solved by the invention]

[0009] Although ipilimumab is effective in treatment, it exhibits dose-limiting toxicities that prevent the administration of higher doses that would be more effective in tumor eradication. There is a need for improved forms of ipilimumab that exhibit a large therapeutic index. Such improved forms of ipilimumab would exhibit enhanced antitumor activity, reduced side effects, or both. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides variants of the anti-human CTLA-4 antibody ipilimumab having mutations in the variable domain that enhance target binding at low / acidic pH (e.g., pH 6.0) compared to binding at neutral pH (e.g., pH 7.4). In particular, the ipilimumab variants of the present invention exhibit preferential binding at low pH, such as pH 5.8, 6.0, 6.2, 6.4, 6.6, or 6.8, relative to neutral pH, such as pH 7.0, 7.2, 7.4, 7.5, or 7.6, compared to ipilimumab. In certain embodiments, preferential binding at low pH is expressed as "acidic pH binding preference" (APBP), which is the ratio of the dissociation equilibrium constant for binding at pH 7.4 to the dissociation equilibrium constant for binding at pH 6.0, i.e., K D-7.4 / K D-6.0 In various embodiments, the APBP is 1.5, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, 35, 50, 75, and 100 or greater. In other embodiments, the preferential binding at low pH is expressed as the "relatively acidic pH binding preference" (CAPBP), which is the ratio of the dissociation equilibrium constant for binding at pH 7.4 to the dissociation equilibrium constant for binding at pH 6.0 for the variant form divided by the corresponding value for ipilimumab, i.e., [(K D-7.4 / K D-6.0 ) バリアント / (K D-7.4 / K D-6.0 ) ipi In various embodiments, the CLPBR is greater than or equal to 1.5, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, 35, 50, 75, and 100. As described herein, the APBP of ipilimumab is essentially 1.0, meaning that the CAPBP is essentially equal to the APBP of the ipilimumab variants disclosed herein.

[0011] In various embodiments, the anti-CTLA-4 antibodies of the invention comprise a heavy chain variable region with histidine (H) substituted at one, two, three, or more non-histidine positions, or with at least one histidine residue in two or more of HCDR1, HCDR2, and HCDR3. In other embodiments, the anti-CTLA-4 antibodies of the invention comprise a light chain variable region comprising an LCDR1 sequence with acidic residues (aspartic acid or glutamic acid; D / E) substituted at one, two, three, or more positions that were not previously aspartic acid or glutamic acid, or with one, two, three, or more aspartic acid or glutamic acid residues in LCDR1. In certain embodiments, the anti-CTLA-4 antibodies of the invention comprise both the heavy and light chain variable regions described above.

[0012] In certain embodiments, the one, two, or three histidine (H) substitutions in the heavy chain variable region are at one or more of residues 31, 56, and 99 of the mature heavy chain sequence. In further embodiments, the one, two, or three acidic (D / E) substitutions in the light chain variable region are at one or more of residues 28, 31, 32, and 33 of the mature light chain sequence. In certain embodiments, the anti-CTLA-4 antibodies of the invention comprise both the heavy and light chain variable regions described above.

[0013] In still further embodiments, improved anti-CTLA-4 antibodies of the invention comprise one, two, or three histidine (H) substitutions at one or more of residues 31, 56, and 99 in the heavy chain variable region of ipilimumab, e.g., as disclosed in SEQ ID NO: 31, or at one or more of residues 30, 31, 52, 53, 54, 56, 101, and 103 in the heavy chain variable region of tremelimumab, e.g., as disclosed in SEQ ID NO: 43. In further embodiments, improved anti-CTLA-4 antibodies of the invention comprise one, two, or three acidic (D / E) substitutions at one or more of residues 28, 31, and 33 in the light chain variable region of ipilimumab, e.g., as disclosed in SEQ ID NO: 32, or at one or more of residues 28, 30, 31, and 32 in the light chain variable region of tremelimumab, e.g., as disclosed in SEQ ID NO: 44. In certain embodiments, the anti-CTLA-4 antibodies of the invention comprise both the heavy and light chain variable regions described above, such as SEQ ID NOs: 31 and 32 or SEQ ID NOs: 43 and 44, but the antibodies of the invention do not comprise both the heavy and light chain variable regions of ipilimumab (SEQ ID NOs: 9 and 10) or tremelimumab (SEQ ID NOs: 39 and 40).

[0014] In various embodiments, ipilimumab variants of the present invention that preferentially bind at low pH include SEQ ID NOs: 15 and 21 (ipi.1); SEQ ID NOs: 16 and 21 (ipi.2); SEQ ID NOs: 17 and 21 (ipi.3); SEQ ID NOs: 18 and 21 (ipi.7); SEQ ID NOs: 11 and 24 (ipi.17); SEQ ID NOs: 11 and 25 (ipi.18); SEQ ID NOs: 11 and 26 (ipi.20); SEQ ID NOs: 11 and 29 (ipi.23); SEQ ID NOs: 11 and 30 (ipi.24); SEQ ID NOs: 11 and 22 (ipi.25); SEQ ID NOs: 11 and 27 (ipi.26); SEQ ID NOs: 12 and 21 (ipi.57); SEQ ID NOs: 19 and 21 (ipi.59); SEQ ID NOs: 12 and 22 (ipi.64); SEQ ID NOs: 19 and 22 (ipi.65); i.66); SEQ ID NOs: 13 and 21 (ipi.69); SEQ ID NOs: 13 and 22 (ipi.71); SEQ ID NOs: 20 and 21 (ipi.82); SEQ ID NOs: 14 and 21 (ipi.84); SEQ ID NOs: 20 and 22 (ipi.86); SEQ ID NOs: 14 and 22 (ipi.88); SEQ ID NOs: 20 and 23 (ipi.90); SEQ ID NOs: 14 and 23 (ipi.92); SEQ ID NOs: 11 and 23 (ipi.93); SEQ ID NOs: 12 and 23 (ipi.94); SEQ ID NOs: 13 and 23 (ipi.95); SEQ ID NOs: 12 and 27 (ipi.100); SEQ ID NOs: 13 and 27 (ipi.101); SEQ ID NOs: 13 and 28 (ipi.105); and SEQ ID NOs: 14 and 28 (ipi.106). In various embodiments, the ipilimumab variants of the present invention that preferentially bind at low pH comprise the heavy and light chain sequences provided above with the addition of a C-terminal lysine (K) residue in the heavy chain.

[0015] In certain embodiments, the ipilimumab variants of the present invention that preferentially bind at low pH are present in an antibody preparation with reduced fucosylation, hypofucosylated antibody preparation, or nonfucosylated antibody preparation. In certain such embodiments, the antibody preparation comprises greater than 95% defucosylated antibody heavy chains. In other embodiments, the antibody preparation comprises 80%-95% defucosylated antibody heavy chains. In yet further embodiments, the antibody preparation exhibits ADCC activity at least twice that of the same antibody preparation produced by conventional means in Chinese hamster ovary (CHO) cells. In yet further embodiments, the antibody preparation is produced in a mammalian cell line deficient in the activity of an enzyme involved in the production of GDP-fucose or its precursor, including, but not limited to, a mammalian cell line partially or completely deficient in alpha-1,6-fucosyltransferase activity, such as a mammalian cell line in which the FUT8 gene has been completely inactivated.

[0016] In another embodiment, the present invention provides nucleic acids encoding the heavy and / or light chains of the pH-sensitive anti-human CTLA-4 antibodies of the present invention, expression vectors containing these nucleic acids, host cells containing these expression vectors, and methods for producing the pH-sensitive anti-human CTLA-4 antibodies of the present invention, comprising culturing the host cells under conditions that allow antibody production and isolating the antibody. The present invention also provides methods for producing the pH-sensitive anti-human CTLA-4 antibodies of the present invention, comprising culturing host cells containing separate expression vectors containing sequences encoding the antibody heavy and light chain sequences, respectively, under conditions that allow the production of the antibody heavy and light chains, and isolating the antibody.

[0017] In another aspect, the present invention provides a method of treating a human subject in need thereof, comprising administering an ipilimumab variant of the present invention. In certain embodiments, the treatment is treatment of cancer, including, but not limited to, unresectable or metastatic melanoma.

[0018] In various embodiments, the treatment is in combination with one or more other anti-tumor therapeutic agents, including other immunomodulators, hi certain embodiments, such immunomodulators are antagonistic antibodies against PD1 or PD-L1.

[0019] In other embodiments, the ipilimumab variants of the invention are administered at a lower or higher dose than the dose approved for the treatment of the same indication for ipilimumab. In one embodiment, the ipilimumab variant is used to treat unresectable or metastatic melanoma and is administered intravenously at a dose of greater than 3 mg / kg, e.g., 10 mg / kg, 20 mg / kg, 50 mg / kg or more, e.g., over 90 minutes q3w for a total of four doses.

[0020] In other embodiments, ipilimumab variants are used as adjuvants in patients with cutaneous melanoma with pathologic involvement of greater than 1 mm of regional lymph nodes who have undergone complete resection, including total lymphadenectomy, and are administered intravenously at a dose of greater than 10 mg / kg, e.g., 20 mg / kg, 50 mg / kg or more, e.g., for a total of 4 doses over 90 minutes q3w followed by q12w for up to 3 years or until documented disease recurrence or unacceptable toxicity.

[0021] In a further embodiment, ipilimumab variants are used in combination with nivolumab to treat patients with intermediate or high risk, previously untreated advanced renal cell carcinoma, administered intravenously at a dose of greater than 1 mg / kg, e.g., 3 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg or more, e.g., over 30 minutes q3w for a total of 4 doses.

[0022] In still further embodiments, ipilimumab variants are used in combination with nivolumab to treat adult and pediatric patients 12 years of age and older with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer that has progressed after treatment with a fluoropyrimidine, oxaliplatin, or irinotecan, at doses greater than 1 mg / kg, e.g., 3 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg or more, e.g., q3w for a total of 4 doses.

[0023] In another embodiment of the invention, the pH-sensitive anti-CTLA-4 antibody is derived from tremelimumab. In various embodiments, the antibody comprises a heavy chain variable region sequence comprising the sequence of SEQ ID NO: 43, wherein a histidine (H) is present at one or more of residues S30, S31, W52, Y53, D54, S56, N57, R101, and A103; a light chain variable region sequence comprising the sequence of SEQ ID NO: 44, wherein an aspartic acid (D) or glutamic acid (E) is present at one or more of residues S28, N30, S31, and Y32; or both. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows the residue numbering for all three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and the first light chain CDR (LCDR1) of ipilimumab. The amino acid sequences are given in single-letter code, conventionally from amino to carboxy terminus, from left to right. The sequences of HCDR1, HCDR2, HCDR3, and LCDR1 are set forth in SEQ ID NOS: 3-6, respectively. The top row of numbers represents the strict sequential numbering used in the sequence listing, which is used to refer to sequence variants in the specification and claims unless otherwise noted. The numbering of residues in the heavy and light chain CDRs follows SEQ ID NOS: 9 and 10, respectively. The bottom row of numbers represents the numbering under the Kabat system, which is used to refer to sequence variants in the figures unless otherwise noted. Omitted numbers in the Kabat numbering are the same as in the sequential numbering. The Kabat numbering system is widely used to facilitate comparison between structurally equivalent residues in antibodies that differ in the number of amino acid residues in the variable domains. The numerical agreement provided in Figure 1 ensures that there is no ambiguity arising from two numbering conventions for the same sequence.

[0025] [Figure 2] Figure 2A shows sequence logos for HCDR1, HCDR2, and HCDR3 (top to bottom) of a pilot library of ipilimumab heavy chain sequence variants. See Example 1. Figure 2B shows sequence logos for these same CDRs after three rounds of selection of phage binding to huCTLA-4 at pH 6.0, but not pH 7.4, as described in Example 1. The single most prevalent sequences for HCDR1, HCDR2, and HCDR3 are T33H, N56H, and T95H, respectively (all numbering according to Kabat). As in all sequence logos herein, the size of the letter representing the amino acid residue at each position is proportional to the relative frequency of that amino acid residue at that position in the library.

[0026] [Figure 3]Figures 3A, 3B, and 3C show the results of electrostatic modeling of ipilimumab bound to human CTLA-4. See Example 1. Figure 3A shows the charge distribution at pH 6, and Figure 3B shows the charge distribution at pH 7.5, with HCDR1 labeled as H1 and LCDR1 labeled as L1. Figure 3C shows a close-up of the electrostatic model at the contact surface between HCDR1 of ipilimumab (e.g., residues T28, S30, S31, and T33 according to Kabat numbering) and human CTLA-4 (e.g., residues E48, D64, and D65).

[0027] [Figure 4] Figures 4A and 4B show sequence logos for the light chain CDR1 (LCDR1) of both the preselected (Figure 4A) and selected combinatorial library (Figure 4B) of ipilimumab sequence variants. The selection protocol to generate the sequences in Figure 4B from the starting set of sequences in Figure 4A is described in Example 2. After selection, a dramatic shift away from the native ipilimumab sequence at positions 27a (S27aE) and 30 (S30E) was observed, compared to other positions where the original ipilimumab residues were favored.

[0028] [Figure 5] Figures 5A and 5B show exemplary surface plasmon resonance affinity data for ipi.25 (LCDR1 S27aE / S30D) at pH 7.4 (Figure 5A) and pH 6.0 (Figure 5B). See Example 2. Antibody ipi.25 binds CTLA-4 5-6 times more tightly at pH 7.4 than at pH 6.0, compared with 1.0 times for ipilimumab. See Table 3 (in Example 2).

[0029] [Figure 6]Figures 6A, 6B, and 6C show the binding parameters at pH 6.0 and pH 7.5 for selected ipilimumab sequence variants of the invention, specifically antibodies ipi.25 (LCDR1 S28E / S31D), ipi.57 (HCDR1 S31H), and ipi.64 (LCDR1 S28E / S31D and HCDR1 S31H). Values were obtained essentially as described in Example 3. Antibody ipi.64 combines the ipi.25 and ipi.57 mutations. The combination of heavy and light chain mutations results in an antibody with enhanced preference for binding at low pH compared to the individual heavy and light chain mutations. The ratios of the dissociation equilibrium binding constants (KD), normalized to the ipilimumab ratio of CAPBP (1.0), are 6.3, 5.5, and 27.2 for ipi.25, ipi.57, and ipi.64, respectively.

[0030] [Figure 7] Figures 7A and 7B show surface plasmon resonance sensorgram data for the binding of ipi.3 (HCDR3 T95H by Kabat numbering) to CTLA-4 at pH 7.4 (Figure 7A) and pH 6.0 (Figure 7B). This mutation was introduced to detune the affinity of ipilimumab at pH 7.5. See Example 4.

[0031] [Figure 8] Figures 8A, 8B, and 8C provide plots of binding of various antibodies of the invention at pH 6.0 and pH 7.4. Figures 8A and 8B show KD values, with lower values representing increased affinity. Antibodies below ipilimumab on these plots have enhanced binding at low pH (pH 6.0). Antibodies toward the lower right quadrant of each plot, based on the position of ipilimumab, also have decreased affinity at pH 7.4, further enhancing their binding preference at low pH (pH 6.0). Figure 8B shows an expanded view of the data from Figure 8A. Figure 8C provides half-life values for complexes of antibodies of the invention with CTLA-4, calculated as ln2 / koff. For this plot, antibodies in the upper left quadrant (also relative to ipilimumab) show enhanced stability at pH 6.0 and reduced stability at pH 7.4. See Example 2.

[0032] [Figure 9] Figure 9 shows the pH dependence of binding of select ipilimumab variants of the invention to CTLA-4. Binding is shown as Req, a measure of steady-state binding in surface plasmon resonance, as a function of pH, with data showing (from left to right) antibodies ipi.64, ipi.71, ipi.92, and ipi.95 at each pH. Ipilimumab data is shown as a line, as indicated in the legend. For all ipilimumab sequence variants, affinity decreases with increasing pH compared to ipilimumab, which binds equally well at all pHs measured. See Example 2.

[0033] [Figure 10] Figures 10A and 10B show binding of various antibodies as Fab fragments to 58α-β-CTLA-4 / CD3ζ cells at pH 7.3 (Figure 10A) and pH 6.0 (Figure 10B). See Example 5. Ipilimumab (IPI fab wild-type, ●) binds equally at both pHs as measured by arbitrary fluorescence intensity units (GMFI) (EC50 of 5.4 nM at pH 7.3 and 7.1 nM at pH 6.0), while the ipilimumab sequence variant ipi.64 (IPI.64 mutant fab, ▲) binds well at pH 6.0 (3.3 nM) and poorly, if at all, at pH 7.3 (140 nM). See Example 5.

[0034] [Figure 11] Figures 11A and 11B show similar results to those shown in Figures 10A and 10B, except that the data are sorted by antibody rather than pH, and data from pH 5.6 have been added. Similar to Figures 10A and 10B, the ipilimumab sequence variant ipi.64 binds much better at pH 6.0 (■) compared to pH 7.3 (●), while ipilimumab binds comparably. See Example 5.

[0035] [Figure 12]Figures 12A, 12B, and 12C show binding of various antibodies of the invention as Fab fragments to 58α-β-CTLA-4 / CD3ζ cells at pH 6.3 (Figure 12A), pH 6.6 (Figure 12B), and pH 7.2 (Figure 12C). As usual, ipilimumab Fab (●) binds equally at all pHs as measured by arbitrary fluorescence intensity units (GMFI), while ipilimumab Fab sequence variants ipi.64 (■) and ipi.71 (▲) show preferential binding at low pH. See Example 5. The curves for ipilimumab Fab sequence variants ipi.64 (■) and ipi.71 (▲) are superimposed in Figure 12C.

[0036] [Figure 13] Figures 13A, 13B, and 13C show similar results to those shown in Figures 12A, 12B, and 12C, except that the data were obtained with full-length IgG antibodies rather than Fab fragments. As expected, the Fab fragments corresponding to the bivalent antibodies bind with higher avidity than the corresponding Fab fragments. Binding of ipilimumab (●), ipi.64 (■), and ipi.71 (▲) is indistinguishable from each other at all pH levels and with similar avidity. See Example 5. Antibodies ipi.92 (▼) and ipi.95 (◆) show preferential binding at low pH in this assay format, consistent with their high KD 7.4 / 6(APBP) values (>59 and 99, respectively) in Table 4.

[0037] [Figure 14] Figures 14A, 14B, and 14C show pH titration of binding of selected antibodies of the invention to human CTLA-4 as measured by surface plasmon resonance spectroscopy, providing the equilibrium binding constant KD, the association / on rate constant kO, and the dissociation / off rate constant kO, respectively. Ipilimumab (●) shows relatively low dependence on pH, while ipi.64 (■), ipi.100 (▲), ipi.101 (▼), ipi.105 (◆), and ipi.106 (○) all show considerable dependence on pH. See Example 8.

[0038] [Figure 15]Figures 15A, 15B, and 15C show pH titration of binding of selected antibodies of the invention to cyno CTLA-4 (CTLA-4 from cynomolgus monkeys (Macaca fascicularis)) as measured by surface plasmon resonance spectroscopy, providing the equilibrium binding constant KD, the association / on rate constant kD, and the dissociation / off rate constant kOFF, respectively. The results are qualitatively similar to those obtained with human CTLA-4 shown in Figures 14A-C, despite the lower affinity. See Example 8.

[0039] [Figure 16] Figures 16A, 16B, and 16C show results similar to those shown in Figures 13A, 13B, and 13C, except that data were obtained for additional antibodies of the invention, all of which are non-fucosylated. See Example 5. Data are shown for ipilimumab (○), ipi.64 (■), ipi.100 (▲), ipi.101 (▼), ipi.106 (● - filled hexagon), and ipi.105 (◆). Antibodies ipi.105 and ipi.106 show preferential binding at low pH.

[0040] [Figure 17]Figures 17A-F show results similar to those shown in Figures 16A-C, except that the data were obtained with a Fab fragment rather than a nonfucosylated full-length IgG1 antibody. See Example 5. Figures 17A and 17B show the results at pH 7.2 at two different magnifications, with Figure 17B showing the data for ipi.106 and ipi.105 enlarged for clarity. Figures 17C and 17D show similar results at pH 6.7, and Figures 17E and 17F show similar results at pH 6.2. Data are shown for ipilimumab (○), ipi.100 (■), ipi.101 (▲), ipi.105 (▼), ipi.106 (◆), and the isotype control (● - filled hexagon). Antibodies ipi.100 and ipi.101 bind as well as ipilimumab at pH 7.2 and progressively better at pH 6.7 and 6.2. Antibodies ipi.105 and ipi.106 bind much worse than ipilimumab at pH 7.2, but show dramatic improvements in binding at pH 6.7 and 6.2, with ipi.105 approaching the affinity of ipilimumab at pH 6.2.

[0041] [Figure 18] Figures 18A, 18B, and 18C show the results of a 58αβ-hCTLA / mCD3ζ:rhB7-1 blocking assay of select antibodies of the invention, expressed as nonfucosylated IgG1, at pH 7.2, 7.0, and 6.8, respectively. Data are shown for ipilimumab (○), ipi.64 (■), ipi.100 (▲), ipi.101 (▼), ipi.105 (◆), ipi.106 (● - filled hexagon), isotype control (□), and no antibody (X). Antibodies ipi.64, ipi.100, and ipi101 were all approximately as effective as ipilimumab at blocking CTLA-4 binding to B7-1, as reflected by suppression of IL-2 secretion, at all pHs, with generally improved blocking at lower pHs. Ipi.105 was less effective than ipilimumab at pH 7.2 but showed roughly equivalent blockage at pH 6.8. Ipi.106 had little, if any, activity at pH 7.2 but moderate activity at pH 6.8. See Example 9.

[0042] [Figure 19]Figures 19A-19M show the effects of select pH-sensitive anti-CTLA-4 antibodies of the present invention, each expressed as a nonfucosylated IgG1, in the MC38 mouse tumor model. Human CTLA-4 knock-in mice were used. See Example 7. Tumor volume (mm3) is provided as a function of days post-implantation, with each line representing one of 10 mice per experiment. Figures 19A, 19B, and 19C show results for ipilimumab administered at 1 mg / kg (mpk), 3 mpk, and 10 mpk, respectively. Figures 19D, 19E, and 19F show similar results at ipi.64. Figures 19G, 19H, and 19I show similar results at ipi.106, and Figures 19J, 19K, and 19L show similar results at ipi.105. Figure 19M shows similar results for mice treated with the isotype control at 10 mpk. Nonfucosylated ipilimumab is effective in inhibiting tumor growth in a dose-dependent manner at 1-10 mpk, with complete inhibition at 3 mpk and 10 mpk. Ipi.64, ipi.106, and ipi.105 are similarly effective in a dose-dependent manner.

[0043] [Figure 20]Figures 20A and 20B show levels of regulatory T cells, expressed as FoxP3+ cells as a percentage of total CD4+ T cells, in the spleen and tumor, respectively, in a mouse tumor model treated with select pH-sensitive anti-CTLA-4 antibodies of the invention. See Example 7. In both figures, circles (●) represent administration of 1 mpk, squares (■) represent 3 mpk, and triangles (▲) represent 10 mpk. Mice were implanted with tumor cells and treated with select antibodies of the invention as described for Figures 19A-19M. Samples from the spleen and tumor were obtained, and the percentage of FoxP3+ cells among total CD4+ T cells was determined by flow cytometry. Nonfucosylated ipilimumab slightly increased Treg levels in the spleen compared to the isotype control, while the pH-sensitive antibodies of the present invention (ipi.64, ipi.106, and ipi.105) only slightly, if at all, increased Treg levels, making them much more similar to the isotype control (Figure 20A). In contrast, nonfucosylated ipilimumab dramatically reduced Treg levels in tumors compared to the isotype control, while the pH-sensitive antibodies of the present invention have the same effect (Figure 20B).

[0044] [Figure 21] Figures 21A and 21B show T cell activation, as measured by the percentage of regulatory T cells that are ICOS+, and proliferation, as measured by the percentage of regulatory T cells that are Ki-67+, in the spleen of a mouse tumor model treated with select pH-sensitive anti-CTLA-4 antibodies of the invention, as described for Figures 20A and 20B. See Example 7. In both figures, circles (●) represent a dose of 1 mpk, squares (■) represent 3 mpk, and triangles (▲) represent 10 mpk. Nonfucosylated pH-sensitive ipilimumab variants show reduced peripheral activity in the spleen, as indicated by the activation and proliferation markers ICOS and Ki-67, compared to nonfucosylated ipilimumab, especially at higher doses where the effect of nonfucosylated ipilimumab is more evident. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description of the Invention definition In order that this disclosure may be more readily understood, some terms will first be defined. As used herein, unless expressly provided otherwise, each of the following terms has the meaning indicated below. Additional definitions are set forth throughout this specification.

[0046] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of antibodies of the invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to a method of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, antibodies of the invention can be administered by topical, epithelial, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical, and other non-parenteral routes. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. Administration can be performed by one or more individuals, including, but not limited to, a doctor, nurse, other medical professional, or the patient themselves.

[0047] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin or antigen-binding portion thereof that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region comprises three domains: C H1 , C H2 and C H3 Each light chain comprises a light chain variable region (herein V L The light chain constant region contains one domain, C L V H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0048] As used herein and in accordance with other common interpretations, an antibody described as comprising a heavy chain and / or a light chain refers to an antibody comprising "at least one" of each heavy chain and / or light chain, and thus includes antibodies having two or more heavy and / or light chains. In particular, antibodies so described encompass conventional antibodies having two substantially identical heavy chains and two substantially identical light chains. Antibody chains can be substantially identical, but not completely identical, if they differ by post-translational modifications such as C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.

[0049] Unless otherwise specified or clear from the context, an antibody defined by target specificity (e.g., an "anti-CTLA-4 antibody") refers to an antibody that can bind to its human target (e.g., human CTLA-4). Such an antibody may or may not bind to CTLA-4 from other species.

[0050] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes can be divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. IgG antibodies, unless otherwise clear from the context, will be referred to herein with the symbol gamma (γ) or simply "G." For example, IgG1 can be represented as "γ1" or "G1." "Isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Unless otherwise specified or clear from the context, the antibodies disclosed herein are human IgG1 antibodies.

[0051] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CTLA-4 is substantially free of antibodies that specifically bind to antigens other than CTLA-4). An isolated antibody that specifically binds to CTLA-4 may, however, cross-react with other antigens, such as CTLA-4 molecules from different species. Furthermore, an isolated antibody is substantially free of other cellular material and / or chemicals. In comparison, an "isolated" nucleic acid refers to a nucleic acid composition that is significantly different from naturally occurring nucleic acids, i.e., has a different chemical identity, properties, and utility. For example, isolated DNA, unlike naturally occurring DNA, is a free-standing portion of naturally occurring DNA and is not an integral part of the larger structural complexes or chromosomes in which it is found in nature. Furthermore, unlike naturally occurring DNA, isolated DNA can be used as a PCR primer or hybridization probe for, among other things, measuring gene expression and detecting biomarker genes or mutations for disease diagnosis or predicting the effectiveness of therapeutic agents. An isolated nucleic acid can also be purified so that it is substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using standard techniques well known in the art.

[0052] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of single molecular composition, i.e., essentially identical in primary sequence and displaying a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0053] As used herein, the term "defucosylated" refers to an individual antibody heavy chain in which the N-linked glycans do not contain fucose residues. As used herein, the term "nonfucosylated" refers to an antibody preparation comprising a defucosylated heavy chain, or, unless otherwise specified, an antibody having greater than 95% defucosylated heavy chains. Such antibody preparations can be used as therapeutic compositions.

[0054] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0055] "Antibody fragment" generally refers to an "antigen-binding portion" of an intact antibody that retains the ability to specifically bind to the antigen bound by the intact antibody ("antigen-binding fragment") or a portion of a whole antibody that includes the Fc region of the antibody that retains FcR binding ability. Exemplary antibody fragments include Fab fragments and single-chain variable domain (scFv) fragments.

[0056] As used herein, "low pH" refers to any pH lower than the normal physiological pH in a typical human subject, such as the pH of normal human blood or serum, e.g., pH 7.4. In a specific embodiment, "low pH" refers to the pH in the tumor microenvironment within a tumor in a human subject treated with an antibody of the present invention, e.g., pH 7.0, 6.8, 6.6, 6.4, 6.2, 6.0, or lower. Unless otherwise specified or clear from the context, normal or physiological pH is pH 7.4, and low pH is pH 6.0. The pH values described are intended to encompass values within the typical error range of the determination of pH in an appropriate sample. In some experiments, pH values of 7.3 and 7.5 were used as surrogates for physiological pH. pH values of 7.4 and 6.0 were selected as exemplary pH values for the experiments described herein to select improved antibodies of the present invention, but such improved antibodies are useful in any situation where the tumor microenvironment has a lower pH than non-tumor tissue. The use of the antibodies of the present invention is in no way limited to any particular pH value used in selection.

[0057] "Acidic pH binding preference" (APBP) is the ratio of the dissociation equilibrium constant for binding at pH 7.4 to the dissociation equilibrium constant for binding at pH 6.0, i.e., K D-7.4 / K D-6.0 Unless otherwise stated or clear from the context, K D The value is e.g. BIACORE (登録商標) The APBP is determined by surface plasmon resonance using a surface plasmon resonance device (GE Healthcare, Chicago, Ill.) or equivalent method. A high APBP represents a greater binding preference at low pH and therefore an improved antibody compared to antibodies with low APBP values. A high APBP can result from enhanced binding at acidic pH with decreased binding at neutral pH, a highly enhanced binding at acidic pH with a slight enhancement of binding at neutral pH, or a slightly decreased binding at acidic pH with a highly decreased binding at neutral pH. Useful antibodies of the present invention can result under any of these scenarios.

[0058] The antibodies of the present invention may exhibit some APBP greater than ipilimumab. To facilitate this comparison, APBP values may be normalized to ipilimumab as the "comparatively acidic pH binding preference" (CAPBP). CAPBP is the ratio of the dissociation equilibrium constant for binding at pH 7.4 to the dissociation equilibrium constant for binding at pH 6.0 of a particular variant of ipilimumab divided by the equivalent value for ipilimumab, i.e., [(K D-7.4 / K D-6.0 ) バリアント / (K D-7.4 / K D-6.0 ) ipi In various embodiments, CAPBP is 1.5, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, 35, 50, 75, and 100 or more.

[0059] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to an in vitro or in vivo cell-mediated reaction in which nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, macrophages, neutrophils, and eosinophils) recognize antibodies bound to surface antigens on target cells, subsequently causing lysis of the target cells. In principle, any effector cell with an activating FcR can be induced to mediate ADCC.

[0060] "Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors or cells that invade nearby tissues and may even metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0061] "Cell surface receptor" refers to molecules and molecular complexes that are capable of receiving signals and transmitting such signals across the plasma membrane of a cell.

[0062] "Effector cell" refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions. Preferably, the cell expresses at least one type of activating Fc receptor, e.g., human FcγRIII, and exerts ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils, and eosinophils.

[0063] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Exemplary "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cellular phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require an Fc region combined with a binding domain (e.g., an antibody variable domain).

[0064] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include the FcγR family of receptors and alternatively spliced forms of these receptors, including allelic variants. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. The various properties of human FcγRs are summarized in Table 1. Most innate effector cell types coexpress one or more activating FcγRs and an inhibitory FcγRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express the inhibitory FcγRIIB in mice or humans.

[0065] "Fc region" (Fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, the Fc region is a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. In IgG, IgA, and IgD antibody isotypes, the Fc region is the second (C1q) of the antibody's two heavy chains. H2 ) and third (C H2 The IgM and IgE Fc regions contain two identical protein fragments derived from heavy chain constant domains (C) in each polypeptide chain; H For IgG, the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as the stretch of amino acid residues from position C226 or P230 to the carboxy terminus of the heavy chain, where numbering is according to the EU index in Kabat. The C of the human IgG Fc region H2 The domain extends from about amino acid 231 to about amino acid 340, while the C H3 The domain is C in the Fc region. H2 The Fc region is located C-terminal to the Fc domain, i.e., extending from about amino acid 341 to about amino acid 447 of IgG. As used herein, the Fc region may be a native sequence Fc or a variant Fc. The Fc may be in the form of an Fc-containing protein polypeptide, such as an isolated Fc region or as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

[0066] [Table 1]

[0067] An "immune response" is a biological response in a vertebrate to foreign agents, which protects the organism from these agents and the diseases they cause. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced either by these cells or by the liver, which result in the selective targeting, binding, damaging, destruction, and / or elimination within the vertebrate body of invading pathogens, cells or tissues infected by pathogens, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0068] "Immunomodulator" or "immunoregulator" refers to a component of a signaling pathway that may be involved in the regulation, control, or modification of an immune response. "Modulation," "regulation," or "modulation" of an immune response refers to any alteration of cells of the immune system or the activity of such cells. Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In a preferred embodiment of the present invention, the immunomodulator is located on the surface of T cells. "Immunomodulatory target" or "immunoregulatory target" refers to an immunomodulator that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by such binding. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0069] "Immunotherapy" refers to treating a subject having or at risk of developing or recurring a disease by methods involving inducing, enhancing, suppressing or otherwise modifying the immune response.

[0070] "Enhancing an endogenous immune response" means increasing the efficacy or potency of an immune response already present in a subject. This increased efficacy and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0071] A "protein" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the chain length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. The term "protein" is used interchangeably with "polypeptide" herein.

[0072] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, rabbits, rodents such as mice, rats, and guinea pigs, avian species such as chickens, amphibians, and reptiles. In preferred embodiments, the subject is a mammal such as a non-human primate, sheep, dog, cat, rabbit, ferret, or rodent. In more preferred embodiments of any of the disclosed inventive aspects, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0073] A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, such as an Fc-fusion protein of the present invention, is any amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom intervals, or prevention of functional impairment or disability due to disease morbidity. A therapeutically effective amount or dosage of a drug also includes a "prophylactically effective amount" or "prophylactically effective dosage," which is any amount of drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing disease or disease recurrence, prevents the onset or recurrence of disease. The ability of a therapeutic agent to promote disease regression or prevent the onset or recurrence of disease can be assessed using a variety of methods known to those skilled in the art, such as assaying the activity of an agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0074] For example, an anti-cancer drug promotes cancer regression in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, prevention of functional or disability impairment due to disease, or other improvement of disease symptoms in a patient. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from drug administration.

[0075] As an example of tumor treatment, a therapeutically effective amount or dosage of a drug preferably inhibits cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to untreated controls. In the most preferred embodiment, a therapeutically effective amount or dosage of a drug completely inhibits cell proliferation or tumor growth, i.e., preferably inhibits cell proliferation or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed in animal model systems, such as the CT26 colon adenocarcinoma, MC38 colon adenocarcinoma, and SalN fibrosarcoma mouse tumor models described herein, which are predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by testing the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro using assays known to those skilled in the art. In other preferred embodiments of the present invention, tumor regression can be observed and sustained for at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

[0076] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent with the purpose of ameliorating, alleviating, ameliorating, arresting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical manifestation associated with a disease.

[0077] Anti-CTLA-4 antibodies with enhanced binding preference at low pH In some embodiments, the present invention provides improved forms of anti-CTLA-4 antibodies, such as ipilimumab, that exhibit enhanced binding preference at low pH, such as in the tumor microenvironment, which may have a pH of 6.5-6.9, compared with 7.2-7.4 in normal tissues. See Figures 14A-14C. Such antibodies are expected to exhibit an improved therapeutic index compared to ipilimumab, i.e., an improved ratio of anti-tumor activity (required for binding in other low-pH compartments of the tumor microenvironment) to side effects (binding mediated by peripheral, non-tumor tissues). Reduced side effects may represent a substantial advantage in light of the level of adverse events observed with anti-CTLA-4 therapy, particularly at high doses. (Ribas et al. (2013) J. Clin. Oncology 31:616; Feng et al. (2013) Clin. Cancer 19:3977).

[0078] Specific K at low and physiological pH D Depending on the value, the improved antibodies of the present invention can be administered at higher, lower, or equivalent doses than those used with ipilimumab. The antibodies of the present invention, all of which necessarily have a CAPBP greater than 1.0, fall into three categories: enhanced affinity at both low and physiological pH (Type I); low affinity at both low and physiological pH (Type II); and enhanced affinity at low pH but low affinity at physiological pH (Type III). Type I antibodies can be administered at lower doses than, for example, ipilimumab while retaining antitumor efficacy. Type III antibodies can be administered at higher doses than, for example, ipilimumab without increasing side effects. Type II antibodies can be administered at lower, higher, or the same doses as, for example, ipilimumab, depending on whether the goal is to maximize safety, maximize efficacy, or balance these two. Nevertheless, the antibodies of the present invention can be administered at any dose that provides the desired efficacy and safety, with the possible dose range being wider than ipilimumab due to the enhanced therapeutic index.

[0079] Other antibodies have been engineered to selectively bind at low pH. Tocilizumab (anti-IL-6R) was modified to selectively reduce binding at pH 6.0, enhancing dissociation from bound IL-6R in acidic endosomes and freeing the antibody to be released into plasma and bind to other IL-6Rs. Igawa et al. (2010) Nat. Biotechnol. 28:1203. Similar approaches have been taken with anti-PCSK-9 antibodies (Chaparro-Riggers et al. (2012) J. Biol. Chem. 287:11090), IL-6 antibodies (Devanaboyina et al. (2013) mAbs 5:851), anti-complement C5 mAb (Fukuzawa et al. (2017) Sci. Reports 7:1080), and the anti-TNFα antibody adalimumab (Schroeter et al. (2015) mAbs 7:138). In each of these cases, unlike the present invention, a relative affinity reduction at low pH compared to neutral / physiological pH was involved to enhance antibody recycling.

[0080] Various human CTLA-4 antibodies with preferential binding affinity at low pH relative to ipilimumab were designed and selected as described in the figures and examples herein. Briefly, a library of nucleic acids was designed to introduce amino acids into the CDRs of ipilimumab to increase the pH sensitivity of binding, and antibodies with the desired property of preferential binding at low pH were selected. The antibody properties and sequences are provided in the sequence listings summarized in Tables 4, 5, 6, 7, and Table 8. The low pH binding preference of the anti-human CTLA-4 antibodies of the present invention can be clearly seen in Figures 8A-8C, Figure 9, and most clearly in Figure 14A, where the antibodies cluster in the quadrant corresponding to preferential binding at low pH on the plot. The antibodies also showed the same trend of low pH binding preference to cyno CTLA-4 (Figure 15A), suggesting that cynomolgus monkeys are a good toxicology model.

[0081] The antibodies of the present invention have been shown to bind to huCTLA-4 when expressed on the cell surface as well as in SPR experiments (Figures 10A-10B, 11A-11B, 12A-12C, 13A-13C, 16A-16C, 17A-17F, and 18A-18C). Select antibodies of the present invention (ipi.64, ipi.106, and ipi.105) were also shown to be as effective as ipilimumab in inhibiting tumor growth in a mouse tumor model using huCTLA-4 knock-in mice when the whole antibody was expressed as a non-fucosylated IgG1 (Figures 19A-19M). These antibodies interact with the tumor microenvironment to the same extent as ipilimumab. reg These same antibodies have been shown to reduce peripheral T cell levels (Figure 20B) and at the same time have a low enhancing effect in the periphery (Figure 20A), suggesting that they may induce fewer side effects in patients known to result from peripheral T cell activation. reg The results also showed lower induction of activation (FIG. 21A) and proliferation (FIG. 21B) in huCTLA-4 cells, consistent with the observation that pH-sensitive anti-huCTLA-4 antibodies of the invention, such as ipi.106 and ipi.105, with or without fucosylation, can maintain the antitumor efficacy of ipilimumab with reduced peripheral toxicity. Such improved antibodies can be administered at higher doses than ipilimumab, where toxicity generally limits administration, to provide enhanced efficacy, or can be administered similarly to ipilimumab with equivalent efficacy but greater safety / tolerability.

[0082] targeted antigen binding In various embodiments, the antibodies of the present invention are modified to selectively block antigen binding in tissues and environments where antigen binding is harmful, while allowing antigen binding where beneficial. In one embodiment, a blocking peptide "mask" is generated that specifically binds to the antigen-binding surface of the antibody and prevents antigen binding, and this mask is linked to each of the antibody's binding arms by a peptidase-cleavable linker. See, e.g., U.S. Patent No. 8,518,404 to CytomX. See also International Patent Publication WO 2018 / 085555. Such constructs are useful for treating cancers in which protease levels are greatly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment releases the masking / blocking peptide, allowing preferential antigen binding in tumors rather than peripheral tissues where antigen binding causes unwanted side effects. Such modifications further enhance the benefits of the low pH preference of the antibodies of the present invention, further enhancing the specificity of ipilimumab activity to tumor sites with low pH and tumor-specific proteases compared to the periphery.

[0083] Alternatively, in a related embodiment, a bivalent binding compound ("masking ligand") is developed that contains two antigen-binding domains that bind to both antigen-binding surfaces of a (bivalent) antibody and prevent antigen binding, where the two binding domain masks are linked to each other (but not to the antibody) by a cleavable linker, e.g., cleavable by a peptidase. See, for example, International Patent Publication WO 2010 / 077643 to Tegopharm Corp. The masking ligand may comprise or be derived from the antigen to which the antibody is intended to bind, or may be produced independently. Such masking ligands are useful for treating cancers in which protease levels are greatly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing the avidity of the antibody for the antigen-binding surface. The resulting dissociation of the masking ligand from the antibody allows preferential antigen binding in tumors over peripheral tissues, where antigen binding causes unwanted side effects.

[0084] Nucleic acid molecules encoding antibodies of the invention Another aspect of the present invention relates to an isolated nucleic acid molecule encoding any of the improved anti-CTLA-4 antibodies of the present invention. The nucleic acid may be present in whole cells, cell lysate, or in a partially purified or substantially pure form. The nucleic acid may be, for example, DNA or RNA, and may or may not contain intronic sequences. In some embodiments, the DNA is genomic DNA, cDNA, or synthetic DNA, i.e., DNA synthesized in a laboratory, for example, by polymerase chain reaction or chemical synthesis.

[0085] pH-sensitive anti-CTLA-4 antibody with enhanced effector function Various modifications to the Fc region of antibodies have been shown to enhance effector function. In mice, enhanced binding to activating Fc gamma receptors and reduced binding to Fc gamma inhibitory receptors follows a hierarchy: mIgG2a >> mIgG2b >> mIgG1-D265A. This hierarchy is defined by Nimmerjahn & Ravetch (2005) Science 310:1510 and follows the activity ratio of immunoglobulin Fc region binding to activating versus inhibitory Fc receptors (known as the A / I ratio) determined for antibodies that mediate ADCC function.

[0086] In some embodiments, the pH-sensitive anti-CTLA-4 antibodies of the invention are human IgG1 antibodies. The ADCC activity of the anti-CTLA-4 antibodies of the invention can be enhanced, for example, by introducing one or more amino acid substitutions in the Fc region, by modifying the glycosylation pattern on N-linked glycans, or both.

[0087] Fc mutations that enhance effector function In certain embodiments, the ADCC activity of the pH-sensitive anti-CTLA-4 antibodies of the present invention is increased by modifying the amino acid sequence of the Fc region, for example, by adding mutations to a naturally occurring human IgG1 sequence to enhance ADCC. With regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2, and the IgG1 constant domain should be selected as the starting point for ADCC enhancement, rather than IgG2 or IgG4. As defined herein, unmodified human IgG1, such as in ipilimumab, does not have enhanced ADCC. The Fc region can be modified by modifying one or more amino acids at the following positions to increase antibody-dependent cellular cytotoxicity (ADCC) and / or increase affinity to Fcγ receptors (FcγR): 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345 6, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. See WO2012 / 142515; see also WO00 / 42072. Exemplary individual substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary clusters of variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. For example, human IgG1 Fcs contains the G236A variant, optionally in combination with I332E, which has been shown to increase the FcγIIA / FcγIIB binding affinity ratio by approximately 15-fold.Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181. Other modifications for enhancing FcyR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. In particular, both ADCC and CDC can be enhanced by altering position E333 of IgG1, e.g., E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. The use of P247I and A339D / Q mutations to enhance effector function in IgG1 is described in WO2006 / 020114, and D280H, K290S±S298D / V is described in WO2004 / 074455. The K326A / W and E333A / S variants have been shown to have effector function in human IgG1, and E333S in IgG2. Idusogie et al. (2001) J. Immunol. 166:2571. Other experiments have shown that G236A / S239D / A330L / I332E results in enhanced binding to FcRIIa and FcRIIIa. Smith et al. (2012) Proc. Nat'l Acad. Sci. (USA) 109:6181; Bournazos et al. (2014) Cell 158:1243.

[0088] Unless otherwise specified or apparent from the context, numbering of amino acid residues in the Fc region of an antibody follows the EU numbering convention (the EU index as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of U.S. Patent Application Publication No. 2008 / 0248028), except when specifically referring to residues in a sequence in a sequence listing, in which case the numbering is necessarily consecutive. For example, references made to the effects of amino acid substitutions in the Fc region generally use EU numbering, which allows reference to a given residue in the Fc region of an antibody with the same number regardless of the length of the variable domain to which it is attached. In rare cases, it may be necessary to refer to the cited literature to confirm the exact Fc residue being referred to.

[0089] In particular, the binding sites of human IgG1 to FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (Shields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII, including the combination mutants T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A (which have enhanced FcγRIIIa binding and ADCC activity). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al. (2006) Proc. Nat'l Acad. Sci. (USA) 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278). Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant showed increased ability to deplete B cells in macaques (Lazar et al. (2006) Proc. Nat'l Acad. Sci. (USA) 103:4005). Furthermore, IgG1 mutants containing the L235V, F243L, R292P, Y300L, V305I, and P396L mutations have been identified that exhibit enhanced binding to FcγRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer.Stavenhagen et al. (2007) Cancer Res. 67:8882; U.S. Patent 8,652,466; Nordstrom et al. (2011) Breast Cancer Res. 13:R123.

[0090] Different IgG isotypes also exhibit differential CDC activity (IgG3>IgG1>>IgG2≒IgG4). Dangl et al. (1988) EMBO J. 7:1989. For uses where enhanced CDC is desired, mutations that increase C1q binding can be introduced. The ability to recruit complement (CDC) can be enhanced by mutations at K326 and / or E333 in IgG2, such as K326W (reduced ADCC activity) and E333S, to increase binding to C1q, the first component of the complement cascade. Idusogie et al. (2001) J. Immunol. 166:2571. Introduction of S267E / H268F / S324T (alone or in any combination) into human IgG1 enhances C1q binding. Moore et al. (2010) mAbs 2:181. The Fc region of the IgG1 / IgG3 hybrid isotype antibody "113F" from Natsume et al. (2008) Cancer Res. 68:3863 (Figure 1 therein) also contributes to enhanced CDC. See also Michaelsen et al. (2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology 93:595.

[0091] Further mutations that can increase or decrease effector function are disclosed in Dall'Acqua et al. (2006) J. Immunol. 177: 1129. See also Carter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol. 20:460.

[0092] Reduced fucosylation, non-fucosylated and hypofucosylated The interaction of the antibodies of the present invention with FcγR can also be enhanced by attaching a modified glycan moiety to each Fc fragment at residue N297. In particular, the absence of core fucose residues strongly enhances ADCC by improving IgG binding to activating FcγRIIIA without altering antigen binding or CD40 activity. (Natsume et al. (2009) Drug Des. Devel. Ther. 3:7). There is compelling evidence that defucosylated tumor-specific antibodies translate into therapeutic activity in in vivo mouse models. (Nimmerjahn & Ravetch (2005) Science 310:1510; Mossner et al. (2010) Blood 115:4393).

[0093] Modification of antibody glycosylation can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Antibodies with reduced or eliminated fucosylation that exhibit enhanced ADCC are particularly useful in the methods of the invention. Cells with altered glycosylation machinery have been described in the literature and can be used as host cells to express the recombinant antibodies of the invention, thereby obtaining antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α-(1,6) fucosyltransferase) (U.S. Patent Application Publication No. 20040110704; Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87: 614), and therefore antibodies expressed in these cell lines lack fucose in the carbohydrate. As another example, EP1176195 describes cell lines in which the FUT8 gene is functionally disrupted and cell lines with little or no activity to add fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells. Shields et al. (2002) J. Biol. Chem. 277:26733. Antibodies with modified glycosylation profiles can also be produced in chicken eggs as described in PCT Publication WO2006 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as duckweed. See, e.g., U.S. Publication 2012 / 0276086. PCT Publication WO99 / 54342 describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit an increase in bisecting GlcNac structures, which increases the ADCC activity of the antibodies. See also Umana et al. (1999) Nat. Biotech. 17:176.Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, the enzyme alpha-L-fucosidase removes fucosyl residues from antibodies. Tarentino et al. (1975) Biochem. 14:5516. Antibodies with reduced fucosylation can also be produced in cells carrying a recombinant gene encoding an enzyme that uses GDP-6-deoxy-D-lyxo-4-hexylose as a substrate, such as GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as described in U.S. Patent 8,642,292. Alternatively, cells can be grown in medium containing a fucose analog that blocks the addition of fucose residues to N-linked glycans or glycoproteins, such as antibodies, produced by cells grown in the medium. U.S. Patent 8,163,551; WO09 / 135181.

[0094] Because defucosylated antibodies exhibit greatly enhanced ADCC compared to fucosylated antibodies, antibody preparations need not be completely free of fucosylated heavy chains to be useful in the methods of the present invention. Residual levels of fucosylated heavy chains do not significantly interfere with the ADCC activity of substantially defucosylated heavy chain preparations. Antibodies produced in conventional CHO cells, which are fully capable of adding core fucose to N-glycans, may nevertheless contain a few percent to up to 15% defucosylated antibodies. Defucosylated antibodies can exhibit 10-fold higher affinity for CD16 and up to 30- to 100-fold enhanced ADCC activity; therefore, even a small increase in the percentage of defucosylated antibodies can dramatically increase the ADCC activity of a preparation. Any preparation containing more defucosylated antibodies than those produced by normal CHO cells in culture may exhibit some degree of enhanced ADCC. Such antibody preparations are referred to herein as preparations with reduced fucosylation. Depending on the original level of defucosylation obtained from normal CHO cells, reduced-fucosylation preparations may contain as little as 50%, 30%, 20%, 10%, or even up to 5% defucosylated antibody. Reduced-fucosylation is defined functionally as a preparation that exhibits a 2-fold or greater enhancement of ADCC compared to antibody prepared with normal CHO cells, and does not refer to any fixed percentage of defucosylated species.

[0095] In other embodiments, the level of defucosylation is defined structurally. As used herein, a nonfucosylated antibody preparation is an antibody preparation containing more than 95%, including 100%, defucosylated antibody heavy chains. A hypofucosylated antibody preparation is an antibody preparation containing heavy chains lacking 95% or less of fucose, for example, an antibody preparation in which 80-95%, e.g., 85-95% and 90-95%, of the heavy chains lack fucose. Unless otherwise specified, hypofucosylated refers to an antibody preparation in which 80-95% of the heavy chains lack fucose, nonfucosylated refers to an antibody preparation in which more than 95% of the heavy chains lack fucose, and "hypofucosylated or nonfucosylated" refers to an antibody preparation in which 80% or more of the heavy chains lack fucose.

[0096] In certain embodiments, hypofucosylated or afucosylated antibodies are produced in cells lacking enzymes essential for fucosylation, such as alpha 1,6-fucosyltransferase encoded by FUT8 (e.g., U.S. Patent No. 7,214,775), or in cells in which exogenous enzymes partially deplete the pool of metabolic precursors for fucosylation (e.g., U.S. Patent No. 8,642,292), or in cells cultured in the presence of small molecule inhibitors of enzymes involved in fucosylation (e.g., WO09 / 135181).

[0097] The level of fucosylation in an antibody preparation can be determined by any method known in the art, including, but not limited to, gel electrophoresis, liquid chromatography, and mass spectrometry. Unless otherwise specified, for purposes of the present invention, the level of fucosylation in an antibody preparation is determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC), essentially as described in Example 3. To determine the level of fucosylation in an antibody preparation, the sample is denatured with PNGase F to cleave N-linked glycans and then analyzed for fucose content. LC / MS of full-length antibody chains is another method for detecting the level of fucosylation in an antibody preparation, although mass spectrometry is inherently less quantitative.

[0098] Pharmaceutical Composition The improved anti-CTLA-4 antibodies of the present invention can comprise a composition, e.g., a pharmaceutical composition, containing the binding protein, e.g., an antibody or fragment thereof, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, subcutaneous, intramuscular, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). The pharmaceutical compositions of the present invention can include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0099] Dosing regimens are adjusted to provide the optimal desired response, e.g., a therapeutic response, or minimal adverse effects. Improved antibodies of the present invention can be administered at the same dose as ipilimumab, but with improved safety. Alternatively, if the antibody exhibits low affinity (or other reduced binding and / or activity) in peripheral tissues, e.g., at a pH of about 7.4, the antibody can be administered at a higher dose than ipilimumab. For metastatic or unresectable melanoma, Yervoy (登録商標) (ipilimumab) will be administered intravenously over 90 minutes at 3 mg / kg every 3 weeks for a total of four doses. (登録商標) (ipilimumab) is administered intravenously at 10 mg / kg. Opdivo, an anti-PD1 antibody, is used for advanced renal cell carcinoma or microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer. (登録商標) (nivolumab) in combination with Yervoy (登録商標) (ipilimumab) is administered intravenously at 1 mg / kg.

[0100] In some embodiments, for example, when the anti-CTLA-4 antibody ipilimumab of the present invention is a type I antibody, it is administered at a lower dose than the approved dose for ipilimumab. Such low administration can exhibit comparable or enhanced anti-tumor efficacy without significantly increasing side effects compared to treatment with unmodified ipilimumab. In an exemplary embodiment, for unresectable or metastatic melanoma, it is administered at less than 3 mg / kg, for example, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or less. In other embodiments, for adjuvant treatment of patients with cutaneous melanoma with pathological involvement of regional lymph nodes greater than 1 mm who have undergone complete resection, including total lymph node dissection, it is administered at less than 10 mg / kg, for example, 5 mg / kg, 3 mg / kg, 1 mg / kg, 0.5 mg / kg or less. The improved ipilimumab variants of the present invention can also be used in combination with at least any combination therapy or bispecific agent in which ipilimumab is used, such as a PD1 or PD-L1 antagonist.

[0101] In certain embodiments, for example when the anti-CTLA-4 antibody ipilimumab of the invention is a Type III antibody, the ipilimumab variant is used to treat unresectable or metastatic melanoma and is administered at a dose of greater than 3 mg / kg, e.g., 10 mg / kg, 20 mg / kg, 50 mg / kg or more. In other embodiments, the Type III ipilimumab variant is an adjuvant for patients with cutaneous melanoma with pathologic involvement of regional lymph nodes greater than 1 mm who have undergone complete resection, including total lymphadenectomy, and is administered at a dose of greater than 10 mg / kg, e.g., 20 mg / kg, 50 mg / kg or more.

[0102] In a further embodiment, type III ipilimumab variants are used in combination with nivolumab to treat patients with intermediate or high-risk, previously untreated advanced renal cell carcinoma, and are administered intravenously at a dose of more than 1 mg / kg, for example, 3 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg or more, for example, for a total of 4 doses over 30 minutes, q3w. In yet a further embodiment, type III ipilimumab variants are used in combination with nivolumab to treat adult and pediatric patients aged 12 years or older with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer that has progressed after treatment with fluoropyrimidine, oxaliplatin or irinotecan, and are administered at a dose of more than 1 mg / kg, for example, 3 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg or more, for example, for a total of 4 doses, q3w.

[0103] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and administration method without being excessively toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field. Those skilled in the art can determine the appropriate dosage based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or administration route selected. The compositions of the present invention can be administered via one or more administration routes using one or more of a variety of methods well known in the art.

[0104] Therapeutic Uses and Methods of the Invention The present invention provides methods for cancer immunotherapy in a subject with cancer, e.g., enhancing the endogenous immune response, thereby treating the subject, comprising administering to the subject a therapeutically effective amount of any of the improved anti-CTLA-4 antibodies described herein.

[0105] In a preferred embodiment of the immunotherapeutic method, the subject is a human.

[0106] Other cancers that may be treated using the immunotherapeutic methods of the present invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Cancers include penile cancer, hematopoietic tumors, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, metastatic cancers, and any combination of said cancers. In a preferred embodiment, the cancer is selected from MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, CRPC, head and neck squamous cell carcinoma, and esophageal, ovarian, gastrointestinal, and breast cancers. The present method is also applicable to the treatment of metastatic cancer.

[0107] Other cancers include hematological malignancies, including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphoblastic lymphoma, and any combination of said cancers.

[0108] Combination therapy In certain embodiments of these methods of treating cancer patients, the improved anti-CTLA-4 antibodies of the present invention are administered to the subject as monotherapy, while in other embodiments, stimulation or blockade of immunoregulatory targets is usefully combined with standard cancer treatments, including chemotherapy regimens, radiation, surgery, hormone ablation, and angiogenesis inhibitors. The improved anti-CTLA-4 antibodies can be conjugated to an anti-neoplastic agent (as an immunoconjugate) or administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapeutic agents. Chemotherapy drugs include, among others, doxorubicin (adriamycin), cyclosporine (cyclosporine), and cyclosporine. (登録商標) ), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (Leukeran (登録商標) ), cyclophosphamide (Cytoxan (登録商標) ;Neosar (登録商標) ), lenalidomide (Revlimid (登録商標) ), bortezomib (Velcade (登録商標) ), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (Treanda) (登録商標) ), rituximab (Rituxan (登録商標) ), ifosfamide, vincristine (Oncovin (登録商標) ), fludarabine (Fludarabine (登録商標) ), thalidomide (thalomid (登録商標) ), alemtuzumab (campus (登録商標) , ofatumumab (Arzera (登録商標) ), everolimus (Afinitor (登録商標) , Zores (登録商標) ) and carfilzomib (Kyprolis TM Co-administration of anti-cancer drugs that work by different mechanisms may help overcome resistance to the drugs or changes in the antigenicity of tumor cells.

[0109] The improved anti-CTLA-4 antibodies of the present invention may be used in combination with other immunomodulatory factors, such as antibodies to other immunomodulatory receptors or their ligands. Several other costimulatory and inhibitory receptors and ligands have been identified that regulate T cell responses. Examples of stimulatory receptors include inducible T cell costimulatory molecule (ICOS), CD137 (4-1BB), CD134 (OX40), CD27, glucocorticoid-inducible TNFR-related protein (GITR) and herpes virus entry mediator (HVEM), while examples of inhibitory receptors include programmed death 1 (PD-1), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), adenosine A2a receptor (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT) and receptor for T cell activation immunoglobulin inhibitory V domain (VISTA). Mellman et al. (2011) Nature 480:480; Pardoll (2012) Nat. Rev. Cancer 12: 252; Baitsch et al. (2012) PloS One 7:e30852. Anti-PD-1 antibody Opdivo (登録商標) (nivolumab) and Keytruda (登録商標)(pembrolizumab) has been approved for use in cancer treatment and can be combined with the improved anti-CLTA-4 antibodies of the present invention. These receptors and their ligands provide targets for therapeutics designed to stimulate or block suppression of the immune response, thereby attacking tumor cells. Weber (2010) Semin. Oncol. 37:430; Flies et al. (2011) Yale J. Biol. Med. 84:409; Mellman et al. (2011) Nature 480:480; Pardoll (2012) Nat. Rev. Cancer 12:252. Stimulatory receptors or receptor ligands are targeted by agonist agents, while inhibitory receptors or receptor ligands are targeted by blocking agents. In particular, the most promising approach to enhancing immunotherapeutic antitumor activity is blockade of so-called "immune checkpoints," which refer to the numerous inhibitory signaling pathways that are important for regulating the immune system, maintaining self-tolerance to minimize collateral tissue damage, and regulating the duration and intensity of physiological immune responses in peripheral tissues. See, e.g., Weber (2010) Semin. Oncol. 37:430; Pardoll (2012) Nat. Rev. Cancer 12:252. Many immune checkpoints are initiated by ligand-receptor interactions and can therefore be easily blocked by antibodies or regulated by recombinant forms of the ligand or receptor.

[0110] This invention is further illustrated by the following examples, which should not be construed as limiting. All figures and all references, patents and published patent applications cited throughout this specification are incorporated by reference in their entirety. quotation and the like, all of which are expressly incorporated herein by reference.

[0111] Example 1 Pilot selection of ipilimumab heavy chain CDR sequence variants with enhanced low pH binding preference In testing a method for selecting pH-optimized sequence variants, one mutation was introduced into each CDR of the heavy chain of ipilimumab, for a total of three amino acid substitutions per heavy chain. The CDRs were modified to histidine (H) at positions 28, 30, 31, 32, and 33 in HCDR1; 52, 52a, 53, 54, 55, and 56 in HCDR2; and 95, 97, and 98 in HCDR3 (all numbering according to Kabat). See Figure 1. The light chain was unchanged.

[0112] Selection of sequence variants with increased binding preference at low / acidic pH was performed essentially as described in Hornsby et al. (2015) Mol. Cell. Proteomics 14:2833. Briefly, ipilimumab light and heavy chain variable domain sequences containing one or more sequence modifications were cloned into an M13 phagemid vector, and phage were generated such that the cloned scFv fragments were expressed on the phage surface as fusions of the encoded proteins. Because the oligonucleotides encoding the ipilimumab light and heavy chain variable domains contained degenerate modifications at selected nucleic acid residues (in this case, a single histidine codon replacing a single native residue in each heavy chain CDR), the resulting phage population contained a library of ipilimumab scFv sequence variants. Nucleic acids encoding the variant ipilimumab light and heavy chain variable domains in this preselection library were sequenced. The distribution of CDR sequences in the library of sequence variants is represented by the sequence logo in Figure 2A.

[0113] This phage library was subjected to four rounds of selection / counterselection for binding / non-binding at pH 6.0 and pH 7.4, respectively. Briefly, biotinylated human CTLA-4 was bound to magnetic streptavidin beads. The phage library was exposed to the beads for 1 hour with gentle agitation at pH 6.0, and the beads were then removed using a magnetic separator, washed at pH 6.0, and eluted at pH 7.4. The phage released at pH 7.4 were then propagated overnight in bacterial cells. The phage obtained from these overnight bacterial cultures were subjected to additional selection rounds similar to the first round above, except that the antigen (huCTLA-4) concentration on the streptavidin magnetic beads was systematically decreased with each subsequent selection round to increase the selection strength.

[0114] The selected phage library obtained after four rounds of selection was used to infect bacteria, which were then plated to allow for clonal selection. Individual colonies were picked, and the nucleic acids encoding the variant ipilimumab light and heavy chain variable domains were sequenced. The distribution of CDR sequences in the resulting collection of sequence variants is represented by the sequence logo in Figure 2B, where the predominant variant for each CDR is represented by a larger letter compared to the largest letter in the preselection library of sequence variants shown in Figure 2A.

[0115] Ipilimumab heavy chain variable domains (Fab constructs) were then generated for each of the predominant variants individually (T33H, N56H, and T95H, according to Kabat numbering) and for all three together (T33H / N56H / T95H), resulting in antibodies designated ipi.1, ipi.2, ipi.3, and ipi.4, respectively. Binding parameters for each of these antibodies to human CTLA-4 are provided in Table 2. Binding parameters were determined by surface plasmon resonance (SPR) as described in Example 3. [Table 2]

[0116] Mutations in HCDR1 and HCDR2 increase binding affinity by 2-3 fold (K D (measured by a decrease in K), whereas mutations in HCDR3 significantly reduced binding, and the combination of all three mutations reduced K D However, all of these results were essentially identical at pH 6.0 and pH 7.4, meaning that although the selection provided antibodies with enhanced affinity (i.e., affinity-matured antibodies), the antibodies did not have an increased binding preference at acidic pH.

[0117] Electrostatic Modeling The electrostatic surface of CTLA4 was modeled using Molecular Operating Environment (MOE) (Chemical Computing Group, Montreal, Canada). The results are shown in Figures 3A, 3B, and 3C. CTLA4 has an electronegative patch consisting of residues E48, D64, and D65 proximal to HCDR1 of ipilimumab (see Figure 3C). Position T33H was enriched in HCDR1 after selection using a combinatorial library. However, it was observed that a single substitution at T33H increased affinity equally at both pH 6.0 and pH 7.4, but did not increase low-pH specificity (Table 2), and therefore was not desirable for further development. Other residues in HCDR1 were enriched with histidines, such as S31, whose side chains are located near the electronegative patch in CTLA4. The S31H variant was generated, yielding ipi.57, which showed binding preference at pH 6.0 compared to pH 7.4 (Table 4). This experiment demonstrates the principle that only sequence variants with an increased ratio of binding at pH 6.0 compared to pH 7.4 are useful in the methods of the invention, rather than any mutations simply due to affinity maturation of the antibody.

[0118] Example 2 A combinatorial library of ipilimumab CDR sequence variants with enhanced low-pH binding preferences After the pilot heavy chain variable screening (Example 1), a fully combinatorial approach was devised to explore modifications in all six CDRs. To obtain sequence variants of ipilimumab with enhanced binding preference at pH 6.0 compared to binding at pH 7.4, mutations were introduced into the heavy and light chain CDRs at selected positions. The CDRs were modified at positions T28, S30, S31, Y32, T33 in HCDR1; F50, S52, Y52a, D53, G54, N55, N56, Y58 in HCDR2; T95, W97, L98 in HCDR3; and Q27, S27a, S30, S31, Y32 in LCDR1; F52, S53, T56 in LCDR2; and Q90, Y91, W96 in LCDR3. Each position was substituted with H (His) and D (Asp) or E (Glu) for LCDR1, with 1-3 mutations per CDR, 1-3 mutations per chain, and a total of 2-6 mutations per scFv. All residue numbering is according to Kabat, as depicted in Figure 1.

[0119] Briefly, oligonucleotides containing the desired mutant ipilimumab heavy and light chain CDR sequences were combined to generate scFv ipilimumab variants with up to three mutations per CDR and one to three mutations in each heavy chain variable domain. These oligonucleotide constructs were cloned into M13 phagemid vectors so that the cloned scFv fragments could be expressed on the phage surface as fusions to the encoded protein.

[0120] Light chain results The pre- and post-selection phage libraries were sequenced to determine which sequences were preferentially enriched in the light chain, regardless of the heavy chain. Exemplary results are shown as sequence logos in Figures 4A and 4B, where the relative abundance of each amino acid residue in LCDR1 is represented by the corresponding single-letter size. While the pre-selection library primarily contains the original ipilimumab residue at the mutation position, the post-selection library clearly shows a high enrichment of different residues at specific positions, such as the LCDR1 mutations S28E and S31E (or S31D) (consecutive numbering). See Figure 4B.

[0121] LCDR1 variants selected at positions 28 and 31 (27a and 30 by Kabat numbering) were tested by surface plasmon resonance and BIACORE (登録商標) On- and off-rate constants (and therefore equilibrium binding constants) were determined at pH 7.4 and pH 6.0 using a surface plasmon resonance spectrometer (Biacore AB, Uppsala, Sweden). See Example 3. Antibodies were tested as monovalent Fab fragments to simplify analysis. Results are provided in Table 3 (top row) for ipi.17 (S28E), ipi.18 (S31D), and ipi.25 (S28E / S31D), and an exemplary sensorgram for ipi.25 is provided in Figures 5A and 5B. Separate experiments were performed to test all pairwise combinations of e-variant sequences at LCDR1 positions 28 and 31 (28E, 28D:31E, 31D; or 27aE, 27aD:30E, 30D in Kabat numbering). Results are shown in Table 3 (bottom row). The combination of S28E and S31D (ipi.25) showed the highest binding preference at pH 6.0 compared to pH 7.4 (K D-7.4 / K D-6.0 =4.0). [Table 3]

[0122] All selected antibody variants were poor binders to CTLA-4 at pH 7.4, with less than a two-fold change in both the on and off rate constants. However, binding at pH 6.0 exhibited similar k a value, but low k d The dissociation equilibrium binding constant (K D ) ratios, normalized to the ratios for ipilimumab in the same experiments, are 1.4, 3.2, and 4.0 for ipi.17, ipi.18, and ipi.25, respectively, in the first experiment, and 5.1, 4.1, 6.3, and 4.3 for ipi.23, ipi.24, ipi.25, and ipi.26, respectively, in the second experiment.

[0123] Characterization of selected variants Binding of select variants to human CTLA-4 was determined essentially as described in Example 3. The results are provided in Table 4. The nomenclature and sequences of these antibodies are provided in Tables 6 and 7 and the sequence listing summarized in Table 8. [Table 4]

[0124] Plots of binding data for select antibodies are provided in Figures 8A-8C. Such plots provide a convenient visual assessment of relative binding preferences at pH 6.0 and pH 7.4. Several antibodies of the invention, such as ipi.64, ipi.71, ipi.92, and ipi.95, showed a dramatic increase in binding preference at pH 6.0 compared to pH 7.4. See Figure 9. All of these antibodies, with light chain 27aE S30D, showed a 5-fold preference (K) for binding at pH 6.0 compared to 7.4. D The LCDR1 S30D (Kabat numbering) variants were selected despite the fact that S27aE had a 3.4-fold preference for S30E, whereas S27aE S30E had only a 3.4-fold preference (Figures 4A and 4B). Nevertheless, the variants ipi.64, ipi.71, ipi.92, and ipi.95, which contain the S30E mutation in LCDR1 rather than the S30D mutation, are provided as ipi.100, ipi.101, ipi.106, and ipi.105, respectively. The reversion of D to E at position 30 (Kabat numbering) in LCDR1 was achieved by excluding the sequence causing unwanted isomerization ("DS").

[0125] Example 3 Surface Plasmon Resonance (SPR) Spectroscopic Determination of Binding Parameters of Antibodies of the Invention Surface plasmon resonance spectroscopy (SPR) was used to determine the binding parameters of various ipilimumab sequence variants of the present invention to human CTLA-4 essentially as follows. Unless otherwise indicated, experiments were performed using BIACORE (登録商標)The assay was performed on an SPR surface plasmon resonance spectrometer (Biacore AB, Uppsala, Sweden). Antibodies were tested as monovalent Fab fragments to simplify the analysis. Data can be presented as sensorgrams such as in Figures 5A, 5B, 7A, and 7B, or as equilibrium dissociation binding constants (K D ), association / on rate constant (k a , k on ), dissociation / off rate constant (k d , k off ) and / or half-life (t 1 / 2 ) can be summarized as coupling parameters such as

[0126] Briefly, the ipilimumab variants of the present invention are produced as Fab fragments and assayed in a BIACORE™ system with an immobilized anti-human kappa polyclonal capture antibody. (登録商標) Capture was performed on a CM4 chip. Monomeric human CTLA-4 was run as an analyte at a maximum concentration of 2 μM. Between cycles, the capture surface was regenerated with 75 mM phosphoric acid. Experiments were performed on a BIACORE chip. (登録商標) The experiments were carried out on a T200 instrument at 37°C. The running buffer was HEPES-buffered saline for the pH 7.4 experiments and Bis-Tris-buffered saline for the low pH experiments. All buffers were supplemented with 0.05% Tween-20 and 1 g / L BSA. The dual-reference sensorgrams were fitted to a 1:1 Langmuir binding model with mass transfer to determine the equilibrium dissociation constant (K D ) and, where appropriate, meetings (k a ) and dissociation (k d ) rate constants were determined.

[0127] Example 4 Mutations that generally reduce affinity A T95H substitution (according to Kabat numbering) in HCDR3 was discovered to detune (generally decrease) the affinity of ipilimumab for CTLA-4, producing antibody ipi.3. Exemplary sensorgrams are provided in Figures 7A (pH 7.4) and 7B (pH 6.0). As can be seen from Table 2, T95H significantly decreased the affinity (K Dincrease) by approximately four-fold, leading to a slight, if any, enhancement of preferential binding at low pH, and the K D-7.4 / K D-6.0 The T95H substitution is 0.9 compared to 1.0. Nevertheless, this mutation was combined with other mutations in various antibodies of the invention that increased binding preference at low pH (i.e., ipi.57, ipi.69, ipi.64, ipi.71, ipi.94, ipi.95, and ipi.105 became ipi.82, ipi.84, ipi.86, ipi.88, ipi.90, ipi.92, and ipi.106, respectively). See Table 7. Without intending to be bound by theory, it was hypothesized that incorporation of the T95H substitution may reduce dose-limiting toxicity (presumably caused by binding at pH 7.4) if such a substitution also affects low pH (in the tumor microenvironment) affinity.

[0128] Example 5 Binding of antibodies of the invention to CTLA-4 on cells Further experiments were performed to confirm that selected antibodies of the present invention bind to the cell surface (and not just in in vitro binding assays) and exhibit enhanced binding preference at low pH. Briefly, antibody ipi.64 was expressed as a Fab fragment and IgG and bound to 58α-β-CTLA-4 / CD3ζ cells in serial dilutions at pH 6.3, 6.6, and 7.2. Cells were incubated with the antibody for 1 hour at 4°C, washed with each buffer, and bound antibody was detected. The results are provided in Figure 10A (pH 7.3) and Figure 10B (pH 6.0). Compared to ipilimumab Fab, antibody ipi.64 exhibited significantly reduced binding at pH 7.3 but enhanced binding at pH 6.0. See Figures 11A and 11B. Such ipilimumab sequence variants are expected to exhibit reduced peripheral binding and therefore far fewer toxic side effects than ipilimumab, while retaining (or improving) affinity and activity in the acidic tumor microenvironment. Such antibodies can be administered at the same dose as ipilimumab (e.g., about 3 mg / kg) with the expectation of enhanced safety, or at higher (more effective) doses (e.g., 10 mg / kg or higher) before dose-limiting toxicity is reached. Ipilimumab is known to be more effective at 10 mg / kg, but is not administered at this level in the setting of unresectable or metastatic melanoma due to toxicity (although it is administered at 10 mg / kg in adjuvant melanoma use).

[0129] Similar experiments were performed to compare binding antibodies ipi.64 and ipi.71 at pH 6.3, 6.6, and 7.2. Results using Fab fragments of the antibodies are provided in Figures 12A, 12B, and 12C, while results with full-length IgG mAbs are provided in Figures 13A, 13B, and 13C.

[0130] As shown in Figures 12A, 12B, and 12C, when the pH-optimized ipilimumab variants were tested in monovalent form as Fab fragments, variants ipi.64 and ipi.71 bound to the same extent as ipilimumab at low pH, but not at high pH.

[0131] In contrast, when the pH-optimized ipilimumab variants were tested in bivalent form as whole antibodies (IgG), as shown in Figures 13A, 13B, and 13C, variants ipi.64 and ipi.71 maintained high affinity at high pH, masking any pH preference, while variants ipi.95 and ipi.92 (among others) showed little binding at high pH.

[0132] Further experiments were performed to determine the pH dependence of binding of the nonfucosylated IgG1 forms ipi.64, ipi.100, ipi.101, ipi.106, and ipi.105 to 58αβ-CTLA4 / mCD3ζ cells. Antibodies were titrated from 100 μg / mL in 1:4 dilutions over 12 points in pH-specific buffers and incubated for 30–60 min. The buffer consisted of HBSS, 2% FBS. HI The solution contained 0.02% sodium azide, 2 mM EDTA, and sodium phosphate buffer (pH 5.4). Sodium phosphate buffer was added until the desired pH was reached (7.2, 6.7, 6.2). Approximately 3e5 cells / well were tested for each cell line. All binding and washing steps were performed with each pH-specific buffer. Detection was performed with a monovalent anti-hkappa secondary nanobody conjugated to Alexa647, used at a 1:2000 dilution. Cells were fixed with BD Fix Solution I for 30 minutes, then resuspended in a pH-specific buffer and read on a cytometer (BD Fortessa). The results are presented in Figures 16A (pH 7.2), 16B (pH 6.7), and 16C (pH 6.2) and show the EC 50 and Cmax values are provided in Table 5. Antibodies ipi.105 and ipi.106 showed increased binding as the pH decreased. [Table 5]

[0133] Further experiments were performed to determine the pH dependence of binding of the Fab forms ipi.100, ipi.101, ipi.106, and ipi.105 to 58αβ-CTLA4 / mCD3ζ cells. The same experimental protocol was used to generate the data in Figures 16A-C. Results are presented in Figures 17A and 17B (pH 7.2), 17C and 17D (pH 6.7), and 17E and 17F (pH 6.2). As with other binding assays, ipilimumab exhibited very similar binding curves regardless of pH. Antibodies ipi.100 and ipi.101 exhibited enhanced binding at low pH, with affinities generally similar to or exceeding those of ipilimumab. Antibodies ipi.105 and ipi.106 also exhibited enhanced binding at low pH, although with lower affinities than ipilimumab.

[0134] Example 6 Antitumor activity of the ipilimumab variants of the present invention in the Sa1N fibrosarcoma tumor model The antitumor activity of the low pH enhanced binding preference ipilimumab sequence variants of the present invention is evaluated in the immunogenic Sa1N fibrosarcoma tumor model. Human CTLA-4 knock-in A / J mice are injected with 2x10 per implant. 6 Sa1N tumor cells are injected subcutaneously. Seven days later, tumors are measured and mice are randomized into treatment groups with comparable mean tumor volumes (e.g., 130-150 mm). 3 / 2). Ipilimumab sequence variants of the present invention and control ipilimumab are administered IP at 200 μg per dose in a 200 μl volume on days 7, 11, and 14. Tumor volume and markers of peripheral anti-CTLA-4 activity are measured 7 days later and at regular intervals until study completion. Peripheral anti-CTLA-4 activity in the mouse tumor model is taken as a surrogate for toxicity in human subjects, as such peripheral activity is responsible for dose-limiting toxicity in patients treated with anti-CTLA-4 antibodies.

[0135] Antibodies that i) reduce tumor growth at least approximately as well as ipilimumab and ii) exhibit lower peripheral anti-CTLA-4 activity than ipilimumab are candidates for the development of improved human therapeutics for any disorder for which ipilimumab is approved or otherwise therapeutically effective.

[0136] Example 7 Antitumor activity of ipilimumab variants of the present invention in the MC38 tumor model The antitumor activity of ipilimumab sequence variants with enhanced low pH binding preference of the present invention was evaluated in the MC38 tumor model as follows.

[0137] Tumor model Briefly, 16-18 week old male and female human CTLA-4 knock-in C57BL / 6 mice were inoculated with 1 × 10 6 MC38 cells were injected subcutaneously. Mice were cultured 8 days after tumor implantation until the tumors reached an average volume of 150 mm 3 Once the mice reached 100 μg / mL, they were randomized into treatment groups. Ipi and pH-sensitive ipi antibodies formulated in PBS were administered intravenously at a single dose of 20 μg, 60 μg, or 200 μg per mouse in each treatment group 9 days after implantation. All anti-CTLA-4 antibodies administered in this example were nonfucosylated IgG1 antibodies. Control mice received 200 μg of an anti-keyhole limpet hemocyanin antibody of the human IgG1-NF isotype. Tumors were measured every 2 to 5 days, and the tumor size was 2000 mm. 3 Mice bearing tumors greater than 100 μg / kg were excluded from the study. 14 days after implantation, treated mice were randomized and sacrificed to select mice for lymphocyte staining analysis. Mice bearing tumors that were not measurable during two or more consecutive measurements were considered tumor-free.

[0138] Figures 19A-19M show that the pH-sensitive antibodies ipi.64, ipi.106 and ipi.105 are as effective as ipilimumab in inhibiting tumor growth in a dose-dependent manner.

[0139] Lymphocyte staining analysis Peripheral anti-CTLA-4 activity in mouse tumor models is taken as a surrogate for toxicity in human subjects, as such peripheral activity is responsible for dose-limiting toxicity in patients treated with anti-CTLA-4 antibodies. Tumor and spleen samples were taken from mice treated with the pH-sensitive ipi antibodies selected above, and the relative activity of the antibodies in the two environments was evaluated as follows.

[0140] Tumors and spleens were harvested from mice 5 days after treatment. Tumors were lysed in gentleMACS C tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) with 250 U / mL collagenase IV (Worthington Biochemical, Lakewood, NJ, USA) and 100 μg / mL DNase I (Sigma-Aldrich, St. Louis, MO, USA) in HBSS supplemented with 5% heat-inactivated FBS (VWR, Radnor Pa., USA) and 5 mM CaCl2 (VWR). Spleens were lysed in gentleMACS C tubes (Miltenyi Biotec) and treated with red blood cell lysis buffer (Sigma-Aldrich). Cells were sieved through a 70-μm filter and resuspended in complete T cell medium (RPMI-1640 supplemented with 10% heat-inactivated FBS, 50 U / mL penicillin / streptomycin, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 2 mM sodium pyruvate, and 10 mM HEPES). Cells were stained with the Zombie NIR fixable viability kit (Biolegend, San Diego, Calif., USA) and treated with TruStain FcX (anti-mouse CD16 / 32) antibody (Biolegend) to block Fc receptor binding. For cell surface staining, cells were stained with CD45 (30-F11, BD Biosciences, San Jose, Calif., USA), CD19 (6D5, Biolegend), CD4 (GK1.5, BD Biosciences), CD8 (53-6.7, Biolegend), and ICOS (7E.17G9, BD Biosciences) in eBioscience flow cytometry staining buffer (ThermoFisher, Waltham Mass., USA).Whole cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher) and stained for FoxP3 (FJK-16s, ThermoFisher), Ki-67 (16A8, Biolegend), and human CTLA4 (BNI3, Biolegend, San Diego, Calif., USA). Samples were analyzed using an LSR Fortessa. (登録商標) Flow data were analyzed using a flow cytometer (BD Biosciences). (登録商標) Flow cytometry data analysis software was used to analyze data using fluorescence-based gates.

[0141] CTLA-4 blockade using anti-CTLA-4 antibodies inhibits T cell proliferation in peripheral sites such as the spleen or lymph nodes. reg and serves as a surrogate for toxicity. Selby et al. (2013) Cancer Immunol. Res. 1:32; Quezada et al. (2006) J. Clin. Invest. 116:1935. Figures 20A and 20B show that the pH-sensitive antibodies ipi.64, ipi.106, and ipi.105 inhibit T cell proliferation in tumors as efficiently as ipilimumab. reg Although it reduces T levels in the periphery (spleen) as much as ipilimumab (Figure 20B), reg Furthermore, Figures 21A and 21B show that these same pH-sensitive anti-CTLA-4 antibodies, compared to ipilimumab, result in less activation as measured by ICOS expression and less T cell proliferation in the periphery (spleen) as measured by Ki-67 expression. reg Figures 21A and 21B show that the IL-16-18 induced less proliferation of IL-16 cells than IL-16 cells. See Figures 21A and 21B, respectively.

[0142] Antibodies that i) reduce tumor growth at least approximately as well as ipilimumab, and ii) exhibit lower peripheral anti-CTLA-4 activity than ipilimumab, as measured by expression of Ki-67 (Selby et al. (2013) Cancer Immunol. Res. 1:32) and ICOS (Liakou et al. (2008) Proc. Nat'l Acad. Sci. (USA) 105:14987), are candidates for the development of improved human therapeutics for any disorder for which ipilimumab is approved or otherwise therapeutically effective. Antibodies ipi.64, ipi.106, and ipi.105 share these advantageous properties.

[0143] Example 8 pH Dependence of Binding Parameters of Ipilimumab Variants of the Invention The pH dependence of binding of selected antibodies of the present invention to human CTLA-4 and cyno CTLA-4 was measured by surface plasmon resonance (SPR) spectroscopy essentially as described in Example 3. Briefly, antibodies were tested as monovalent Fab fragments. An anti-human kappa polyclonal capture antibody (Southern Biotech, Ala., USA) was immobilized on an ethylenediamine-blocked CM4 sensor chip of a Biacore T200 surface plasmon resonance system. Fab fragments of selected antibodies of the present invention were then captured in one flow cell each. Monomeric human CTLA-4 or cyno CTLA-4 was run as analytes at nominal concentrations of 2 μM, 400 nM, 80 nM, and 16 nM in each buffer condition. The running buffer was 20 mM BisTris, 150 mM NaCl, 0.05% Tween-20, 1 g / L BSA, with a pH ranging from 5.5 to 7.4 in 0.1-0.2 pH unit intervals (12 total). The surface was regenerated between 1 / 200 H3PO4 cycles. All data were double-referenced and fitted to a 1:1 Langmuir binding model with transport limitations using Biacore T200 Evaluation software. If necessary, R max was fixed by calculating the binding expectancy based on the capture level and apparent activity from others of the same Fab.

[0144] The results for human CTLA-4 are shown in Figure 14A(K D ), 14B(k on ) and 14C(k off ) is provided in Figure 14A. Figure 14A shows the binding affinity (K) of the Fab fragments of various antibodies of the invention as a function of pH. D ) Ipilimumab, as expected, shows little change as a function of pH. Antibodies ipi.64, ipi.100, ipi.101, ipi.105, and ipi.106 all show a systematic decrease in affinity with increasing pH. Ipi.105 shows the greatest difference in affinity at pH 5.5 and 7.5, with affinity generally higher than ipilimumab. Ipi.106 also shows a roughly equivalent change in affinity with pH, but with much lower affinity across the entire range, with affinity lower than ipilimumab at pH 6.5 and above.

[0145] Figures 14B and 14C show the rate constants (k) for the association / on and dissociation / off rates of the same Fab fragment as a function of pH. on and k off ) as a function of pH. As with the overall affinity, ipilimumab shows little change in either constant as a function of pH. In contrast, all of the antibodies of the present invention show a significant decrease in k on shows a similar decrease in k off The pH dependence of the antibodies ipi.105 and especially ipi.106 is substantially different, with the antibodies ipi.105 and ipi.106 exhibiting k off These kinetic results show a dramatic increase in K at ipi.105 and ipi.106 compared to ipi.64, ipi.100, and ipi.101. D The large dependence of k on pH off It is shown that this is driven by an increase in

[0146] Similar results for cyno CTLA-4 are shown in Figure 15A(K D ), 15B(k on ) and 15C(k off) The results are qualitatively similar to those observed with human CTLA-4, but with generally lower affinity. Figure 14A shows the binding of various Fab fragments of the antibodies of the invention as a function of pH, primarily the k off The increase in binding affinity (K D ) The similar pH dependence of binding of human and cyno CTLA-4 indicates that cynomolgus monkeys are good animals for toxicology and other studies using the antibodies of the invention. See Example 10.

[0147] Example 9 Biological Activity of Ipilimumab Variants of the Invention in the 58αβ-hCTLA / mCD3ζ:rhB7-1 Blocking Assay The biological activity of select pH-sensitive ipilimumab variants of the present invention was evaluated in a 58αβ-hCTLA4 / mCD3ζ:rhB7-1 blocking assay as follows. One day before the assay, rhB7-1 was diluted in 1x PBS and plated at 1.2 μg / mL in 96-well flat-bottom plates at 50 μL / well and incubated overnight at 4°C. Ipilimumab pH variants (Ipi.64, Ipi.100, Ipi.101, Ipi.105, Ipi.106) were expressed as IgG1-NF variants and tested for blocking 58αβ-hCTLA4 / mCD3ζ cells and plate-bound rhB7-1. Ipi-pH variants were titrated in pH-specific media at a final maximum concentration of 15 μg / mL in eight 1:4 dilutions. The media was RPMI-1640, 10% FBS. HI The coated plate contained sodium bicarbonate and sodium phosphate buffer (pH 5.4). Sodium phosphate buffer was added until the desired pH was reached (7.2, 7.0, 6.8). 120 μL of cells at 1e6 cells / mL and 60 μL of antibody titration were combined in a 96-well round-bottom plate and incubated at 37°C, 8% CO2 for 20 minutes to allow the pH to equilibrate. The coated plate was washed with PBS, and 150 μL of the cell / antibody mixture was transferred to the plate. The plate was incubated at 37°C, 8% CO2 for 18 hours, and the supernatant was harvested and tested for mIL-2 production by ELISA (BD). The results are presented in Figures 18A-18C.

[0148] Example 10 Toxicity evaluation in cynomolgus monkeys A 4-week study in accordance with the Good Laboratory Practice Regulations for nonclinical Laboratory Studies of the US Food and Drug Administration (21 CFR Part 58), the USDA Animal Welfare Act (9 CFR, Parts 1, 2, 3), and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (ILAR publication 1996) will be conducted in cynomolgus monkeys to evaluate the toxicity of the pH-sensitive ipilimumab variants of the invention compared to ipilimumab.

[0149] Thirty purpose-bred cynomolgus monkeys (Macaca fascicularis; 5 monkeys / sex / group) were assigned to three groups using a stratified randomization scheme designed to achieve similar group mean body weights. Groups were randomly assigned to treatments. Groups received 1) saline control, 2) ipilimumab 50 mg / kg, or 3) a pH-sensitive ipilimumab variant of the present invention 50 mg / kg intravenously (IV) once weekly (on days 1, 8, 15, and 22) for a total of four doses. Animals were evaluated for clinical signs and changes in body weight, and cardiovascular evaluations were performed. (Selby et al. (2016) PLoS One 11:e0167251). [Table 6] *- Further variants ipi.23 and ipi.24, containing ipi HC (SEQ ID NO: 11) and LC var. 9 (SEQ ID NO: 29) or LC var. 10 (SEQ ID NO: 30), respectively, are not included in Table 6 solely for space reasons, but are included in Table 7.

[0150] [Table 7]

[0151] [Table 8-1] [Table 8-2]

[0152] For antibody sequences, the sequence listing provides the sequences of the mature variable regions of the heavy and light chains, i.e., the sequences do not include the signal peptide. Any signal sequence suitable for use with the production cell line being used can be used to produce the antibodies of the present invention. The heavy chain amino acid sequences are shown without the C-terminal lysine residue, although in certain embodiments, such a residue is encoded in the nucleic acid construct for the antibody. Numbering in the specification and figures often follows the Kabat numbering system and therefore may not correspond to the numbering in the sequence listing, although a comparison with Figure 1 will clear up any ambiguity.

[0153] Embodiments of the present invention: Embodiments of the present invention are further described in the following sections: [Section 1] An anti-human CTLA-4 antibody or antigen-binding fragment thereof having an acidic pH binding preference (APBP) of 1.5, 1.5, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, 35, 50, 75 or 100 or more bindings at acidic pH compared to neutral pH. [Section 2] The anti-human CTLA-4 antibody or fragment according to item 1, wherein the acidic pH is 5.8, 6.0, 6.2, 6.4, 6.6 or 6.8, and the neutral pH is 7.0, 7.2, 7.4, 7.5 or 7.6. [Section 3] The anti-human CTLA-4 antibody or fragment according to item 2 above, which has an acidic pH of 6.0 and a neutral pH of 7.4. [Section 4] A heavy chain variable region sequence comprising an HCDR1, HCDR2 and HCDR3 sequence, wherein: a. HCDR1 contains at least two histidine (H) residues; b. HCDR2 contains at least one histidine (H) residue; and / or c. HCDR3 contains at least one histidine (H) residue; The anti-human CTLA-4 antibody or fragment according to any one of items 1 to 3 above. [Section 5] 5. The anti-human CTLA-4 antibody or fragment according to any one of items 1 to 4, comprising a light chain variable region sequence including an LCDR1 sequence containing one, two, or three acidic residues, each of which is selected from the group consisting of aspartic acid (D) and glutamic acid (E). [Section 6] The anti-human CTLA-4 antibody or fragment according to any one of items 4 to 5 above, comprising a heavy chain variable region sequence containing one or more residues selected from the group consisting of 31H, 55H and 95H (Kabat numbering). [Section 7] 7. The anti-human CTLA-4 antibody or fragment according to any one of items 4 to 6, comprising a light chain variable region sequence comprising one or more residues selected from the group consisting of 27aD, 27aE, 30D, 30E, 31D, 31E, 32D and 32E (Kabat numbering). [Section 8] 4. The anti-human CTLA-4 antibody or fragment of any of items 1 to 3 above, comprising a heavy chain variable region sequence comprising the sequence of SEQ ID NO: 31, wherein a histidine (H) is present at one or more of residues 31, 56 and 99. [Section 9] The anti-human CTLA-4 antibody or fragment of any of paragraphs 1 to 3 and 8, comprising a light chain variable region sequence comprising the sequence of SEQ ID NO: 32, wherein an aspartic acid (D) or glutamic acid (E) is present at one or more of residues 28, 31 and 33. [Section 10] 10. The anti-human CTLA-4 antibody or fragment of any of items 8 to 9 above, comprising a heavy chain variable region sequence comprising the sequence of SEQ ID NO: 31, wherein histidine (H) is present at two or more of residues 31, 56 and 99. [Section 11] 11. The anti-human CTLA-4 antibody or fragment of any of items 8 to 10 above, comprising a light chain variable region sequence comprising the sequence of SEQ ID NO: 32, wherein aspartic acid (D) or glutamic acid (E) is present at two or more of residues 28, 31 and 33. [Section 12] 12. The anti-human CTLA-4 antibody or fragment of any of items 8 to 11, comprising a light chain variable region sequence comprising the sequence of SEQ ID NO: 32, wherein an aspartic acid (D) or a glutamic acid (E) is present at each of residues 28, 31, and 33. [Section 13] 13. The anti-human CTLA-4 antibody or fragment thereof according to any one of items 8 to 12 above, comprising a heavy chain variable region sequence comprising the sequence of SEQ ID NO:14. [Section 14] a) residues 1 to 118 of SEQ ID NO: 15 and residues 1 to 108 of SEQ ID NO: 21 (ipi.1); b) residues 1 to 118 of SEQ ID NO: 16 and residues 1 to 108 of SEQ ID NO: 21 (ipi.2); c) residues 1 to 118 of SEQ ID NO: 17 and residues 1 to 108 of SEQ ID NO: 21 (ipi.3); d) residues 1 to 118 of SEQ ID NO: 18 and residues 1 to 108 of SEQ ID NO: 21 (ipi.7); e) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 24 (ipi.17); f) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 25 (ipi.18); g) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 26 (ipi.20); h) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 29 (ipi.23); i) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 30 (ipi.24); j) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 22 (ipi.25); k) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 27 (ipi.26); l) residues 1 to 118 of SEQ ID NO: 12 and residues 1 to 108 of SEQ ID NO: 21 (ipi.57); m) residues 1 to 118 of SEQ ID NO: 19 and residues 1 to 108 of SEQ ID NO: 21 (ipi.59); n) residues 1 to 118 of SEQ ID NO: 12 and residues 1 to 108 of SEQ ID NO: 22 (ipi.64); o) residues 1 to 118 of SEQ ID NO: 19 and residues 1 to 108 of SEQ ID NO: 22 (ipi.66); p) residues 1 to 118 of SEQ ID NO: 13 and residues 1 to 108 of SEQ ID NO: 21 (ipi.69); q) residues 1-118 of SEQ ID NO: 13 and residues 1-108 of SEQ ID NO: 22 (ipi.71); r) residues 1 to 118 of SEQ ID NO: 20 and residues 1 to 108 of SEQ ID NO: 21 (ipi.82); s) residues 1 to 118 of SEQ ID NO: 14 and residues 1 to 108 of SEQ ID NO: 21 (ipi.84); t) residues 1 to 118 of SEQ ID NO: 20 and residues 1 to 108 of SEQ ID NO: 22 (ipi.86); u) residues 1 to 118 of SEQ ID NO: 14 and residues 1 to 108 of SEQ ID NO: 22 (ipi.88); v) residues 1 to 118 of SEQ ID NO: 20 and residues 1 to 108 of SEQ ID NO: 23 (ipi.90); w) residues 1 to 118 of SEQ ID NO: 14 and residues 1 to 108 of SEQ ID NO: 23 (ipi.92); x) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 23 (ipi.93); y) residues 1 to 118 of SEQ ID NO: 12 and residues 1 to 108 of SEQ ID NO: 23 (ipi.94); z) residues 1 to 118 of SEQ ID NO: 13 and residues 1 to 108 of SEQ ID NO: 23 (ipi. 95); aa) residues 1 to 118 of SEQ ID NO: 12 and residues 1 to 108 of SEQ ID NO: 27 (ipi.100); bb) residues 1-118 of SEQ ID NO: 13 and residues 1-108 of SEQ ID NO: 27 (ipi.101); cc) residues 1 to 118 of SEQ ID NO: 13 and residues 1 to 108 (ipi.105) of SEQ ID NO: 28; and dd) residues 1 to 118 of SEQ ID NO: 14 and residues 1 to 108 (ipi.106) of SEQ ID NO: 28 4. The anti-human CTLA-4 antibody or fragment thereof according to any one of items 1 to 3 above, comprising a pair of heavy chain variable region sequence and light chain variable region sequence selected from the group consisting of: [Section 15] a) SEQ ID NOs: 15 and 21 (ipi.1); b) SEQ ID NOs: 16 and 21 (ipi.2); c) SEQ ID NOs: 17 and 21 (ipi.3); d) SEQ ID NOs: 18 and 21 (ipi.7); e) SEQ ID NOs: 11 and 24 (ipi.17); f) SEQ ID NOs: 11 and 25 (ipi.18); g) SEQ ID NOs: 11 and 26 (ipi.20); h) SEQ ID NOs: 11 and 29 (ipi.23); i) SEQ ID NOs: 11 and 30 (ipi.24); j) SEQ ID NOs: 11 and 22 (ipi.25); k) SEQ ID NOs: 11 and 27 (ipi.26); l) SEQ ID NOs: 12 and 21 (ipi.57); m) SEQ ID NOs: 19 and 21 (ipi.59); n) SEQ ID NOs: 12 and 22 (ipi.64); o) SEQ ID NOs: 19 and 22 (ipi.66); p) SEQ ID NOs: 13 and 21 (ipi.69); q) SEQ ID NOs: 13 and 22 (ipi.71); r) SEQ ID NOs: 20 and 21 (ipi.82); s) SEQ ID NOs: 14 and 21 (ipi.84); t) SEQ ID NOs: 20 and 22 (ipi.86); u) SEQ ID NOs: 14 and 22 (ipi.88); v) SEQ ID NOs: 20 and 23 (ipi.90); w) SEQ ID NOs: 14 and 23 (ipi.92); x) SEQ ID NOs: 11 and 23 (ipi.93); y) SEQ ID NOs: 12 and 23 (ipi.94); z) SEQ ID NOs: 13 and 23 (ipi.95); aa) SEQ ID NOs: 12 and 27 (ipi.100); bb) SEQ ID NOs: 13 and 27 (ipi.101); cc) SEQ ID NOs: 13 and 28 (ipi.105); and dd) SEQ ID NOs: 14 and 28 (ipi.106) Item 15. The anti-human CTLA-4 antibody of item 14, comprising a pair of heavy and light chain sequences selected from the group consisting of: [Section 16] 8 (ipi.105), an anti-human CTLA-4 antibody or fragment of item 15 above, comprising the heavy and light chain sequences of SEQ ID NOs: 13 and 2. [Section 17] 16. The anti-human CTLA-4 antibody or fragment of item 15, comprising the heavy and light chain sequences of SEQ ID NOs: 14 and 28 (ipi.106). [Section 18] 18. A preparation of an anti-human CTLA-4 antibody according to any one of items 1 to 17, wherein the anti-human CTLA-4 antibody preparation has reduced fucosylation, is hypofucosylated, or is non-fucosylated. [Section 19] 19. The anti-human CTLA-4 antibody preparation of paragraph 18, wherein the antibody is produced in a mammalian cell line lacking alpha-1,6-fucosyltransferase activity. [Section 20] A nucleic acid encoding the heavy chain and / or light chain of the anti-human CTLA-4 antibody or fragment thereof according to any one of items 1 to 17 above. [Section 21] An expression vector comprising the nucleic acid of Item 20 above. [Section 22] A host cell comprising the expression vector of item 21 above. [Section 23] A method for producing an anti-human CTLA-4 antibody, comprising culturing the host cell of item 22 under conditions that allow the production of the anti-human CTLA-4 antibody, and isolating the anti-human CTLA-4 antibody. [Section 24] A method for treating a disease in a human subject, comprising administering to the subject a therapeutic amount of any one of the antibodies or fragments thereof or anti-human CTLA-4 antibody preparations of items 1 to 19 above. [Section 25] 25. The method according to item 24, wherein the disease is cancer. [Section 26] 26. The method of any of items 24 to 25, wherein the treatment or treatment is carried out in combination with an antagonistic antibody or fragment thereof that binds to PD1 or PD-L1. [Section 27] 27. The method according to any one of items 25 to 26, wherein the cancer is unresectable or metastatic melanoma. [Section 28] 28. The method of paragraph 27, wherein the antibody is administered at a dose of more than 3 mg / kg, for example 10 mg / kg, 20 mg / kg, 50 mg / kg or more. [Section 29] 27. The method of any of items 25 to 26, wherein the cancer is melanoma and the antibody is administered as an adjuvant to a patient with cutaneous melanoma with pathologic involvement of more than 1 mm of regional lymph nodes who has undergone complete resection including total lymph node dissection. [Section 30] 30. The method of paragraph 29, wherein the antibody is administered at a dose of more than 10 mg / kg, for example 20 mg / kg, 50 mg / kg or more. Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An anti-human CTLA-4 antibody or antigen-binding fragment thereof, having an acidic pH binding preference (APBP) of 1.5 or greater for binding at acidic pH compared to neutral pH, f) residues 1-118 of SEQ ID NO:11 and residues 1-108 of SEQ ID NO:25 (ipi.18); g) residues 1-118 of SEQ ID NO:11 and residues 1-108 of SEQ ID NO:26 (ipi.20); h) residues 1-118 of SEQ ID NO:11 and residues 1-108 of SEQ ID NO:29 (ipi.23); i) residues 1-118 of SEQ ID NO:11 and residues 1-108 of SEQ ID NO:30 (ipi.24); j) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 22 (ipi.25); k) residues 1 to 118 of SEQ ID NO: 11 and residues 1 to 108 of SEQ ID NO: 27 (ipi.26); l) residues 1-118 of SEQ ID NO:12 and residues 1-108 of SEQ ID NO:21 (ipi.57); m) residues 1-118 of SEQ ID NO:19 and residues 1-108 of SEQ ID NO:21 (ipi.59); n) residues 1-118 of SEQ ID NO:12 and residues 1-108 of SEQ ID NO:22 (ipi.64); o) residues 1-118 of SEQ ID NO:19 and residues 1-108 of SEQ ID NO:22 (ipi.66); p) residues 1 to 118 of SEQ ID NO: 13 and residues 1 to 108 of SEQ ID NO: 21 (ipi.69); q) residues 1-118 of SEQ ID NO:13 and residues 1-108 of SEQ ID NO:22 (ipi.71); r) residues 1-118 of SEQ ID NO:20 and residues 1-108 of SEQ ID NO:21 (ipi.82); s) residues 1-118 of SEQ ID NO:14 and residues 1-108 of SEQ ID NO:21 (ipi.84); t) residues 1-118 of SEQ ID NO:20 and residues 1-108 of SEQ ID NO:22 (ipi.86); u) residues 1-118 of SEQ ID NO:14 and residues 1-108 of SEQ ID NO:22 (ipi.88); v) residues 1-118 of SEQ ID NO:20 and residues 1-108 of SEQ ID NO:23 (ipi.90); w) residues 1-118 of SEQ ID NO:14 and residues 1-108 of SEQ ID NO:23 (ipi.92); x) residues 1-118 of SEQ ID NO:11 and residues 1-108 of SEQ ID NO:23 (ipi.93); y) residues 1-118 of SEQ ID NO:12 and residues 1-108 of SEQ ID NO:23 (ipi.94); z) residues 1-118 of SEQ ID NO:13 and residues 1-108 of SEQ ID NO:23 (ipi.95); aa) residues 1 to 118 of SEQ ID NO: 12 and residues 1 to 108 of SEQ ID NO: 27 (ipi.100); bb) residues 1-118 of SEQ ID NO:13 and residues 1-108 of SEQ ID NO:27 (ipi.101); cc) residues 1-118 of SEQ ID NO:13 and residues 1-108 (ipi.105) of SEQ ID NO:28; and dd) residues 1 to 118 of SEQ ID NO: 14 and residues 1 to 108 (ipi.106) of SEQ ID NO: 28 a heavy chain variable region sequence pair and a light chain variable region sequence pair selected from the group consisting of: wherein the acidic pH is 6.0 and the neutral pH is 7.4; An anti-human CTLA-4 antibody or antigen-binding fragment thereof.

2. f) SEQ ID NOs: 11 and 25 (ipi.18); g) SEQ ID NOs: 11 and 26 (ipi.20); h) SEQ ID NOs: 11 and 29 (ipi.23); i) SEQ ID NOs: 11 and 30 (ipi.24); j) SEQ ID NOs: 11 and 22 (ipi.25); k) SEQ ID NOs: 11 and 27 (ipi.26); l) SEQ ID NOs: 12 and 21 (ipi.57); m) SEQ ID NOs: 19 and 21 (ipi.59); n) SEQ ID NOs: 12 and 22 (ipi.64); o) SEQ ID NOs: 19 and 22 (ipi.66); p) SEQ ID NOs: 13 and 21 (ipi.69); q) SEQ ID NOs: 13 and 22 (ipi.71); r) SEQ ID NOs: 20 and 21 (ipi.82); s) SEQ ID NOs: 14 and 21 (ipi.84); t) SEQ ID NOs: 20 and 22 (ipi.86); u) SEQ ID NOs: 14 and 22 (ipi.88); v) SEQ ID NOs: 20 and 23 (ipi.90); w) SEQ ID NOs: 14 and 23 (ipi.92); x) SEQ ID NOs: 11 and 23 (ipi.93); y) SEQ ID NOs: 12 and 23 (ipi.94); z) SEQ ID NOs: 13 and 23 (ipi.95); aa) SEQ ID NOs: 12 and 27 (ipi.100); bb) SEQ ID NOs: 13 and 27 (ipi.101); cc) SEQ ID NOs: 13 and 28 (ipi.105); and dd) SEQ ID NOs: 14 and 28 (ipi.106) The anti-human CTLA-4 antibody of claim 1, comprising a heavy chain sequence and a light chain sequence pair selected from the group consisting of:

3. The anti-human CTLA-4 antibody of claim 2, comprising the heavy and light chain sequences of SEQ ID NOs: 13 and 28 (ipi.105).

4. The anti-human CTLA-4 antibody of claim 3, comprising the heavy and light chain sequences of SEQ ID NOs: 14 and 28 (ipi.106).

5. The anti-human CTLA-4 antibody according to any one of claims 1 to 4, wherein the antibody has reduced fucosylation, is hypofucosylated, or is non-fucosylated.

6. The anti-human CTLA-4 antibody of claim 5, wherein the antibody is produced in a mammalian cell line lacking alpha-1,6-fucosyltransferase activity.

7. A nucleic acid encoding the heavy and light chains of the anti-human CTLA-4 antibody or antigen-binding fragment of any one of claims 1 to 4.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. A method for producing an anti-human CTLA-4 antibody, comprising culturing the host cell of claim 9 under conditions that allow the production of the anti-human CTLA-4 antibody, and isolating the anti-human CTLA-4 antibody.

11. A pharmaceutical composition for treating a disease in a human subject, comprising a therapeutic amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

12. The pharmaceutical composition of claim 11, wherein the disease is cancer.

13. The pharmaceutical composition of claim 11 or 12, wherein the treatment is carried out in combination with an antagonistic antibody or antigen-binding fragment thereof that binds to PD1 or PD-L1.

14. The pharmaceutical composition of claim 12 or 13, wherein the cancer is unresectable or metastatic melanoma.

15. 15. The pharmaceutical composition of claim 14, wherein the antibody is administered at a dose greater than 3 mg / kg.

16. 14. The pharmaceutical composition of claim 12 or 13, wherein the cancer is melanoma and the antibody is administered as an adjuvant to patients with cutaneous melanoma with pathologic involvement of regional lymph nodes greater than 1 mm who have undergone complete resection including total lymphadenectomy.

17. 17. The pharmaceutical composition of claim 16, wherein the antibody is administered at a dose greater than 10 mg / kg.

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