PH-sensitive anti-CTLA-4 antibodies and uses thereof

Abstract

Provided herein are pH sensitive anti-CTLA-4 antibodies, including variants of irpimumab, and therapeutic uses thereof.

Description

Technical Field

[0001] This invention relates to pH-sensitive anti-CTLA-4 antibodies (including variants of ipilimumab) and their therapeutic uses.

[0002] References to sequence lists

[0003] This application contains a sequence list, which was submitted via the Patent Centre in XML format and is incorporated herein by reference in its entirety. The file was created on May 7, 2024, named Sequence_Listing_111005_0605_01PC00.xml, and is 42 kb in size. Background Technology

[0004] Two major challenges of targeting CTLA-4 in cancer immunotherapy are reducing immune-related adverse events (irAEs) and improving broad therapeutic efficacy. Conventionally, anti-CTLA-4 antibody-triggered tumor immunity and irAEs are considered inseparable. CTLA-4 checkpoint blockade enhances the tumor-killing response, which inevitably leads to cross-reactive autoimmunity, making irAEs a necessary price to pay for successful anti-cancer immunotherapy.

[0005] Commercial anti-CTLA-4 monoclonal antibody (mAb) ipilimumab is frequently associated with a wide variety of adverse events in clinical monotherapy or combination therapy. In particular, combination therapy with ipilimumab and nivolumab (anti-PD-1) results in over 50% of patients with both melanoma and NSCLC experiencing grade 3 and 4 irAEs. Ipilimumab-related irAEs include hematological abnormalities such as pure red cell aplasia and non-infectious inflammatory damage to solid organs. While some low-grade irAEs such as colitis or dermatitis are manageable, high-grade irAEs such as pneumonia, hepatitis, neurological events, and myocarditis can be serious and life-threatening. Although patients who survive three years with ipilimumab do not show a further decline in ten-year survival compared to those treated with anti-PD-1 (which illustrates the specific benefits of ipilimumab for immunotherapy), irAEs remain a major threat to treated patients. It not only prevented many patients from continuing immunotherapy, but also limited the efficacy of CITE.

[0006] Recent research has provided a new perspective on these challenges. Cancer immunotherapeutic effects (CITE) and irAEs have been demonstrated to represent different activities of anti-CTLA-4 antibodies. The former is due to the selective reduction of regulatory T cells (Tregs) in the tumor microenvironment, while the latter can be attributed to the loss of CTLA-4 on the cell surface. Specifically, the intracellular pH-sensitive binding sensitivity of anti-CTLA-4 antibodies to CTLA-4 has been shown to be key to controlling irAEs. pH-insensitive versions of antibodies allow for sustained binding to CTLA-4 after antibody-induced endocytosis, which triggers lysosomal degradation of CTLA-4, thereby causing irAEs. In contrast, antibodies with lower binding affinity at low pH detach from CTLA-4 during transport through low-pH compartments, allowing CTLA-4 to recycle back to the plasma membrane, thus preventing irAEs. More importantly, pH-sensitive antibodies that allow CTLA-4 to recirculate to the cell surface of Tregs within the tumor microenvironment (TME) trigger stronger antibody-dependent cytotoxicity / antibody-dependent phagocytosis (ADCC / ADCP) due to higher target density, leading to more effective attenuation of Tregs and better CITE in the tumor. This new understanding makes it possible to improve CTLA-4 targeting and uncouple irAEs from CITE by altering the protein sequence of antibodies to change their pH sensitivity. Ipilimumab is a pH-insensitive antibody, which contributes to its tendency to induce irAEs. Therefore, there is a need in the art for pH-sensitive ipilimumab variants with a reduced risk of inducing irAEs. Summary of the Invention

[0007] This document provides anti-CTLA antibodies. Anti-CTLA antibodies may include a light chain variable region and a heavy chain variable region. The light chain variable region may include a complementarity determination region (CDR) 1 containing the sequence shown in SEQ ID NO: 1, a CDR2 containing the sequence shown in SEQ ID NO: 3, and a CDR3 containing the sequence shown in SEQ ID NO: 5; and the heavy chain variable region may include a CDR1 containing the sequence shown in SEQ ID NO: 7, a CDR2 containing the sequence shown in SEQ ID NO: 8, and a CDR3 containing the sequence shown in SEQ ID NO: 12. The light chain variable region may include a CDR1 containing the sequence shown in SEQ ID NO: 2, a CDR2 containing the sequence shown in SEQ ID NO: 3, and a CDR3 containing the sequence shown in SEQ ID NO: 4; and the heavy chain may include a CDR1 containing the sequence shown in SEQ ID NO: 6, a CDR2 containing the sequence shown in SEQ ID NO: 10, and a CDR3 containing the sequence shown in SEQ ID NO: 12. The light chain variable region may include CDR1 containing the sequence shown in SEQ ID NO: 2, CDR2 containing the sequence shown in SEQ ID NO: 3, and CDR3 containing the sequence shown in SEQ ID NO: 4; and the heavy chain variable region may include CDR1 containing the sequence shown in SEQ ID NO: 6, CDR2 containing the sequence shown in SEQ ID NO: 11, and CDR3 containing the sequence shown in SEQ ID NO: 12.

[0008] The light chain variable region may contain the sequence shown in SEQ ID NO: 16, and the heavy chain variable region may contain the sequence shown in SEQ ID NO: 28. The light chain variable region may contain the sequence shown in SEQ ID NO: 15, and the heavy chain variable region may contain the sequence shown in SEQ ID NO: 26. The light chain variable region may contain the sequence shown in SEQ ID NO: 15, and the heavy chain variable region may contain the sequence shown in SEQ ID NO: 27.

[0009] Anti-CTLA-4 antibodies may comprise a light chain and a heavy chain. The light chain may comprise the sequence shown in SEQ ID NO: 21, and the heavy chain may comprise the sequence shown in SEQ ID NO: 35. The light chain may comprise the sequence shown in SEQ ID NO: 20, and the heavy chain may comprise the sequence shown in SEQ ID NO: 33. The light chain may comprise the sequence shown in SEQ ID NO: 20, and the heavy chain may comprise the sequence shown in SEQ ID NO: 34.

[0010] Compared to ipilimumab, anti-CTLA-4 antibodies can bind to CTLA-4 with reduced affinity at a pH of approximately 5.5–6.0.

[0011] This document also provides compositions comprising an anti-CTLA-4 antibody and pharmaceutically acceptable excipients. The composition may comprise 20 mM histidine buffer, 8.8% (w / v) α,α-trehalose dihydrate, 0.06% (w / v) PS80, and 0.2 mM EDTA•2Na•2H2O. The composition may have a pH of about 6.0. The composition may further comprise an anti-PD-1 antibody. The anti-PD-1 antibody may comprise a light chain comprising the sequence shown in SEQ ID NO: 36 and a heavy chain comprising the sequence shown in SEQ ID NO: 37.

[0012] This article provides methods for treating cancer in subjects with this need, which may include administering an anti-CTLA-4 antibody or composition. This article also provides anti-CTLA-4 antibodies or compositions for treating cancer, and their use in the preparation of medicaments for treating cancer. Cancers may include melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), or triple-negative breast cancer (TNBC). Cancers may be solid tumors. Anti-CTLA-4 antibodies or compositions may be administered intravenously or are intended to be administered intravenously. Up to 10 mg / kg of anti-CTLA-4 antibody may be administered to subjects, or is intended to be administered to subjects. Administration may be once every 3 weeks or is intended to be once every 3 weeks. Attached Figure Description

[0013] This patent or application document contains at least one drawing in color. Upon request and payment of the necessary fees, the Patent Office will provide a copy of this patent or application disclosure with the color drawings.

[0014] Figure 1The design of pH-sensitive ipilimumab variants is shown, where the complementarity-determining region (CDR) includes histidine substitutions of tyrosine residues. 23 variants include different combinations of Y-to-H substitutions in the CDR. LH00 (light chain VLM0 and heavy chain LHM0): wild-type ipilimumab. The light chain variable regions VLM0-VLM3 have SEQ ID NO: 14-17, and the heavy chain variable regions VHM0-VHM5 have SEQ ID NO: 23-28, respectively.

[0015] Figure 2A -D shows the analysis of pH-sensitive ipilimumab variants generated by introducing histidine-to-tyrosine substitution in the complementarity-determining region (CDR). His-hCTLA-4 (0.5 μg / ml) was coated in ELISA plates, and different anti-CTLA4-mAbs were added at 1 μg / ml in buffers ranging from pH 4.5 to 7.0. Antibody binding to CTLA-4 was measured using horseradish peroxidase-labeled anti-human IgG antibodies. pH binding of CTLA-4 between ipilimumab variants and wild-type antibodies is shown in different mutant combinations. Figure 2A In each of the -D variants, the ipilimumab variant comprises various combinations of Y to H substitutions in at least one of the heavy chain and light chain variable regions. In each of these, VHM0-VHM5 respectively have SEQ ID NO: 23-28. Figure 2A The mutant with substitutions only in the heavy chain variable region is shown. VLM0 has SEQ ID NO: 14. Figure 2B The mutant with Y-to-H substitution in both Y regions of the light chain variable region is shown. VLM3 has SEQ ID NO: 17. Figure 2C The mutant with Y to H substitutions only in CDR1 of the light chain is shown. VLM1 has SEQ ID NO: 15. Figure 2D The mutant with Y to H substitutions only in the CDR3 region of the light chain variable region is shown. VLM2 has SEQ ID NO: 16.

[0016] Figure 3A -B shows the pH binding sensitivity of ipilimumab variants LH13, LH14, and LH25 compared to the wild-type (LH00) and pH-sensitive antibody HL12. The method was performed as described in Figure 2. Figure 3A The original data is displayed. Figure 3B The data are shown relative to pH 7. LH13: (light chain Y33H; heavy chain Y59H); LH14: (light chain Y33H; heavy chain Y58H); LH25: (light chain Y92H; heavy chain Y32H).

[0017] Figure 4The results showed that the pH-6 sensitive ipilimumab variant, rather than the pH-6 insensitive variant, dissociated from CTLA-4 during antibody-induced internalization. Stable 293T cell lines expressing hCTLA-4 were labeled with anti-CTLA-4 mAb at 4°C for 30 min. After washing out unbound antibody, cells were transferred to 37°C for 1 h. Antibody-bound surface CTLA-4 was captured by protein-G beads and detected by Western blot.

[0018] Figure 5 Surface plasmon resonance (SPR) measurements of affinity for ipilimumab variants at different pH values ​​were presented. Binding affinity of ipilimumab, LH13, and LH25 to his-CTLA-4 was assessed by SPR analysis using a Biacore T100 biosensor (GE Healthcare). 1000 response units (RUs) of protein A (Sigma-Aldrich) from Staphylococcus aureus were immobilized on flow cells 1 and 2 of a Series S Sensor ChipCM5 (GE, BR100530). Approximately 150 RUs of antibody were directly captured on flow cell 2. Binding experiments were performed at 25°C in 10 mM HEPES, 150 mM NaCl, and 0.05% (v / v) Tween 20 at pH 7.4 or pH 6. The his-CTLA-4 was used with a double titration series (0.46875 nM-60 nM for pH 7.4 / 0.976 nM-60 nM for pH 6). Between runs, the sensor surface was regenerated with two 45-second injections of 20 mM HCl. Sensing plots were used for dual reference against the control flow cell (Fc1) and the three buffer injections.

[0019] Figure 6 The binding effects of ipilimumab and its pH-sensitive variants on cell surface CTLA-4 were demonstrated. Stable 293T cell lines expressing hCTLA-4 were treated with ipilimumab and its variants LH13 and LH25 at 37°C for 4 hours, and then plasma membrane proteins of the treated cells were separated using the MINUTE™ Plasma Membrane Protein Separation and Cell Grading Kit (SM-005, Invent Biotechnologies, INC.). Cell surface CTLA-4 was detected by Western blotting using CTLA-4 antibodies (H-126, sc-9094). Tubulin and Na+ were also analyzed. + / K + ATPase α1 is used as a marker for cytoplasmic fractionation and plasma membrane fractionation, respectively.

[0020] Figure 7 This shows the pH-6 insensitive ipilimumab variant, rather than the pH-6 sensitive variant, entering lysosomes after internalization. Ipilimumab WT, LH13, or LH25 cells were labeled with AF488 and treated with a stable CHO cell line expressing hCTLA-4 at 4°C. After washing away excess antibody, the cells were incubated at 37°C for 30 min and further stained with lysotracker. Colocalization between AF488-labeled anti-CTLA-4 mAb and lysosomes is shown by confocal images (green: anti-CTLA-4 mAb; magenta: lysosomes; white: overlap of green and magenta). Scale bars: 10 μm.

[0021] Figure 8 This demonstrates the use of pH-6 sensitive ipilimumab variants, rather than pH-6 insensitive variants, to rescue CTLA-4 from lysosomal degradation. Ipilimumab WT, LH13, or LH25 cells were labeled with AF488 and treated at 4°C with stable CHO cell lines expressing hCTLA-4-OPF. After washing away additional antibody, cells were incubated at 37°C for 30 min and further stained with lysotracker. Colocalization of AF488-labeled anti-CTLA-4 mAb, lysosomes, and CTLA-4 tagged with orange fluorescent protein (OFP) is shown in representative confocal images (green: anti-CTLA-4 mAb; red: CTLA-4; blue: lysosomes; white: overlap of the three markers). Scale bars: 10 μm.

[0022] Figure 9A -B shows weight loss and anemia induced by pH-6 sensitive ipilimumab variant salvage combination therapy. Figure 9A C57BL / 6 Ctla4h / h mice were treated on days 10, 13, 16, and 19 with control human IgG-Fc + anti-PD-1, ipilimumab + anti-PD-1, LH13 + anti-PD-1, and LH25 + anti-PD-1 at a dose of 100 μg / mouse / injection, respectively. Major growth retardation was measured every three days. One mouse from the human IgG-Fc + anti-PD-1 treatment group and two mice from the ipilimumab + anti-PD-1 and LH13 + anti-PD-1 treatment groups were excluded from the analysis due to death before the experimental endpoint; these mice exhibited severe growth retardation. Figure 9B CBC analysis was performed on day 39 postnatally. To avoid cage variation, mice in the same cage were individually tagged and treated with different antibodies. Data are presented as mean ± SEM, with n = 22–24 mice per group. The tests were performed double-blindly. Samples were collected from three independent experiments. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni multiple comparison test. P < 0.001; P < 0.0001.

[0023] Figure 10A -B shows multi-organ inflammation induced by pH-6 sensitive ipilimumab variant salvage combination therapy. Analysis Figure 9A The same mice used in the -B experiment. Autopsy was performed on day 40 after birth. Figure 10A Representative images of H&E-stained paraffin sections from different organs. Representative inflammatory lesions are marked with arrows. Scale bars, 200 μm. Figure 10B The toxicity scores of viscera and glands were assessed. Samples were collected from three independent experiments and were scored in a double-blind manner. Data were analyzed using one-way ANOVA and Bonferroni multiple comparison test. P < 0.0001.

[0024] Figure 11A -B demonstrates the immunotherapeutic effects of ipilimumab and its pH-sensitive variants. Figure 11A Tumor growth in mice receiving hIgG-Fc, ipilimumab, LH13, or LH25 in YM3.3 melanoma tumors (left panel) or MC38 colon tumors (right panel) is shown. In the YM3.3 melanoma model, mice (n=13-14) were treated with three doses of antibody at 1.5 mg / kg / dose on days 10, 13, and 16 post-tumor inoculation. The same treatment was performed in the MC38 colon cancer model (n=12-13) on days 17 and 20 post-tumor inoculation with two doses of antibody. Figure 11B Showing Figure 11A Kaplan-Meier survival curves of tumor-bearing mice. Data are mean ± SEM and are from two independent experiments. Statistical analysis was performed using two-way repeated measures ANOVA and Bonferroni multiple comparison test. P < 0.05; P < 0.01; P < 0.001; P < 0.0001.

[0025] Figure 12 The study demonstrated Treg reduction of ipilimumab and its pH-sensitive variant in the tumor microenvironment and spleen. On day 14 post-tumor inoculation, Ctla4 mice carrying YM3.3 were treated with control hIgG, ipilimumab, LH13, or LH25 (100 μg / mouse). h / hMice (n = 7). Treg reduction in the tumor microenvironment (left panel) and spleen (right panel) was determined by the percentage of Treg cells in CD4 T cells 16 h after antibody treatment. Data are mean ± SEM and are from two independent experiments. Statistical tests were performed using two-way repeated measures ANOVA and Bonferroni multiple comparison test. P < 0.05; P < 0.01. Detailed Implementation

[0026] The inventors have discovered a pH-sensitive ipilimumab variant that unexpectedly eliminates irAE while improving its CITE.

[0027] 1. Definition.

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. Unless the context clearly requires otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural indicators.

[0029] In describing the ranges of numbers in this article, each intermediate number with the same precision is explicitly considered. For example, for the range of 6–9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range of 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0030] As used herein, the term "antibody" refers to an immunoglobulin molecule that has an antigen recognition site called a "variable region." The term "variable region" refers to a domain in an immunoglobulin that is distinct from the widely shared domains of antibodies (such as the antibody Fc domain). Variable regions contain "hypervariable regions," whose residues are responsible for antigen binding. The hypervariable region contains amino acid residues from the “complementarity-determining region” or “CDR” (i.e., typically around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and around residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Ref. 44), and may also contain residues from the “hypervariable ring” (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain). The “frame region” or “FR” residues are those variable domain residues other than those in the hypervariable region as defined herein. The antibodies disclosed herein may be monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camel-like antibodies, single-chain antibodies, disulfide-linked Fv (sdFv), intrabody, or anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-anti-Id antibodies against the antibodies of the present invention). In particular, antibodies may be immunoglobulin molecules such as IgG, IgE, IgM, IgD, IgA, or IgY, or belong to classes such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, or subclasses.

[0031] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s “variable region” antigen recognition site and exhibit the ability to bind antigens with immunological specificity, containing the antibody’s complementarity-determining region (“CDR”) and optionally framework residues. Such fragments include Fab’, F(ab’)2, Fv, single-chain (ScFv) and their mutants, naturally occurring variants, and fusion proteins that contain the antibody’s “variable region” antigen recognition site with a heterologous protein (e.g., toxins, antigen recognition sites of different antigens, enzymes, receptors or receptor ligands, etc.). As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acids, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0032] Human antibodies, chimeric antibodies, or humanized antibodies are particularly preferred for use in humans; however, mouse antibodies or antibodies from other species can be advantageously used for many purposes (e.g., in vitro or in situ detection assays, acute in vivo use, etc.).

[0033] "Chimeric antibodies" are molecules in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a constant region of a human immunoglobulin. Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR transplantation (EP 239,400; International Publication No. WO 91 / 09967; and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;46-48), and chain tampering (US Patent No. 5,565,332), the contents of which are incorporated herein by reference.

[0034] The antibodies described herein may be humanized antibodies. As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more core receptors (CDRs) derived from a non-human (typically mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "receptor." A constant region is not required, but if present, it must be substantially identical to the constant region of a human immunoglobulin, i.e., at least about 85%–90%, preferably about 95% or higher. Thus, all portions of the humanized immunoglobulin (possibly except for the CDR) are substantially identical to the corresponding portions of the native human immunoglobulin sequence. Humanized antibodies are antibodies comprising humanized light chains and humanized heavy chains of immunoglobulins. For example, humanized antibodies will not encompass typical chimeric antibodies, as the entire variable region of, for example, a chimeric antibody is non-human. Donor antibodies are referred to as "humanized" through a "humanization" process because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody providing the CDR. Humanized antibodies can be human immunoglobulins (receptor antibodies), wherein hypervariable residues of the receptor are replaced by hypervariable residues from non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates with the desired specificity, affinity, and ability. In some cases, framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues not found in either the receptor or donor antibodies. These modifications can further improve antibody performance. Humanized antibodies can contain at least one, typically two, substantially entire variable domains, wherein all or substantially all of the hypervariable regions correspond to the hypervariable regions of non-human immunoglobulins, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies may also optionally contain at least a portion of the immunoglobulin constant region (Fc), which can be the constant region of human immunoglobulins that binds immunospecifically to the FcγRIIB peptide, and this constant region has been altered by introducing one or more amino acid residues through substitution, deletion, or addition (i.e., mutation).

[0035] 2. pH-sensitive ipilimumab variant

[0036] This article provides information on anti-CTLA-4 antibodies and their antigen-binding fragments. Anti-CTLA-4 antibodies can bind specifically to CTLA-4, particularly human CTLA-4. CTLA-4 can be expressed on the surface of living cells at endogenous or transfected concentrations. Living cells can be T cells, which can be regulatory T cells (Tregs). Antibodies can be monoclonal antibodies, human antibodies, chimeric antibodies, or humanized antibodies. Antibodies can also be monospecific, bispecific, trispecific, or multispecific. Antibodies can be detectably labeled and can contain conjugated toxins, drugs, receptors, enzymes, or receptor ligands. Anti-CTLA-4 antibodies can induce cancer rejection while reducing immune-related adverse effects (irAEs) associated with immunotherapy. Anti-CTLA-4 antibodies can effectively induce Fc receptor-dependent Treg attenuation and tumor rejection. Anti-CTLA-4 antibodies can dissociate from CTLA-4 under acidic pH conditions within cells, which allows CTLA-4 to recycle back to the cell surface.

[0037] Anti-CTLA-4 antibodies can be variants of ipilimumab. Compared to ipilimumab, anti-CTLA-4 antibodies can be pH-sensitive and can exhibit reduced binding to CTLA-4 at acidic pH conditions. The pH at which pH-sensitive anti-CTLA-4 antibodies exhibit reduced binding to CTLA-4 can be approximately 4.5, 5.0, 5.5, 6.0, or 6.5. The pH can be 4.5–6.0, and particularly approximately 5.5 or 6.0. In one instance, the pH at which anti-CTLA-4 antibodies exhibit reduced binding affinity to CTLA-4 can be approximately 6.0.

[0038] Compared to wild-type ipilimumab, the amino acid sequence of the variant may contain one or more histidine substitutions for tyrosine near or within one or more complementarity-determining regions (CDRs) of at least one heavy or light chain. The CDRs may be one or more of CDR1, CDR2, and CDR3. Anti-CTLA-4 antibodies may contain a light chain variable region, which may contain one or more of the following CDRs.

[0039] Table 1. Light chain variable region CDR sequences

[0040]

[0041] The variable region of the light chain may contain CDR1, CDR2 and CDR3, which respectively contain the sequences shown in the table below.

[0042] Table 2. Combinations of CDRs in the Variable Region of Light Chains

[0043]

[0044] Anti-CTLA-4 antibodies may contain a heavy chain variable region, which may contain one or more of the following CDRs.

[0045] Table 3 Heavy chain variable region CDR sequences

[0046]

[0047] The heavy chain variable region can contain CDR1, CDR2, and CDR3, which respectively contain the sequences shown in the table below.

[0048] Table 4 Heavy chain variable region CDR combinations

[0049]

[0050] The variable region of a light chain can contain one of the following sequences.

[0051] VLM0

[0052] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 14)

[0053] VLM1

[0054] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSHLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 15)

[0055] VLM2

[0056] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHGSSPWTFGQGTKVEIK (SEQ ID NO: 16)

[0057] VLM3

[0058] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSHLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHGSSPWTFGQGTKVEIK (SEQ ID NO: 17)

[0059] The light chain may include a constant region comprising the following sequence.

[0060] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)

[0061] The light chain may contain one of the following sequences.

[0062] VLM0

[0063] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 19)

[0064] VLM1

[0065] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSHLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 20)

[0066] VLM2

[0067] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 21)

[0068] VLM3

[0069] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSHLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 22)

[0070] The heavy chain variable region may contain one of the following sequences.

[0071] VHM0

[0072] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 23)

[0073] VHM1 [[ID=]19]

[0074] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHTMHWVRQAPGKGLEWVTFISYDGNNKHHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDHWGQGTLVTVSS (SEQ ID NO: 24)

[0075] VHM2

[0076] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDHWGQGTLVTVSS (SEQ ID NO: 25)

[0077] VHM3

[0078] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 26)

[0079] VHM4

[0080] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 27)

[0081] VHM5

[0082] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 28)

[0083] Anti-CTLA-4 antibodies may include a heavy chain constant region derived from a human Ig protein, which may be IgG, IgE, IgM, IgD, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In one example, the constant region is the Fc region derived from the human IgG1 protein. The heavy chain may include a constant region containing the following sequences.

[0084] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29)

[0085] The heavy chain constant region may comprise the following sequence.

[0086] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 40)

[0087] The heavy chain constant region may comprise the following sequence.

[0088] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38)

[0089] The C-terminal lysine (K) can be additionally included in the amino acid sequence of the heavy chain shown in SEQ ID NO: 38, which can increase expression levels. The terminal lysine can be cleaved naturally during the production of the anti-CTLA-4 antibody or at the time of antibody administration.

[0090] The heavy chain constant region may also contain one or more mutations. Relative to the sequence shown in SEQ ID NO: 29, 38, or 40, one or more mutations may be selected from M135Y, S137T, T139E, S181A, E216A, and K217A, and combinations thereof. In one example, the heavy chain constant region of the antibody contains all six mutations. The mutated heavy chain constant region may contain the following sequences.

[0091] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 39)

[0092] The heavy chain may comprise one of the following sequences.

[0093] VHM0

[0094] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 30)

[0095] VHM1

[0096] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHTMHWVRQAPGKGLEWVTFISYDGNNKHHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 31)

[0097] VHM2

[0098] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 32)

[0099] VHM3

[0100] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 33)

[0101] VHM4

[0102] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 34)

[0103] VHM5

[0104] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 35)

[0105] Anti-CTLA-4 antibodies may comprise the following combinations of light chain variable regions and heavy chain variable regions: the light chain variable region having a combination of CDRs 1-3 as shown in Table 2, and the heavy chain variable region having a combination of CDRs 1-3 as shown in Table 4. In one example, the light chain variable region is VLM0 and the heavy chain variable region is VLM0, referred to herein as LH00. In another example, the light chain variable region is VLM0 and the heavy chain variable region is VHM1, referred to herein as LH01. Anti-CTLA-4 antibodies may contain a combination of light chain variable regions and heavy chain variable regions selected from LH01, LH02, LH03, LH04, LH05, LH10, ​​LH11, LH12, LH13, LH14, LH15, LH20, LH21, LH22, LH23, LH24, LH25, LH30, LH31, LH32, LH33, LH34, and LH35. Anti-CTLA-4 antibodies may contain one of the following combinations of light chain variable regions and heavy chain variable regions.

[0106] Table 5

[0107]

[0108] Anti-CTLA-4 antibodies may contain a combination of light and heavy chains selected from LH01, LH02, LH03, LH04, LH05, LH10, ​​LH11, LH12, LH13, LH14, LH15, LH20, LH21, LH22, LH23, LH24, LH25, LH30, LH31, LH32, LH33, LH34, and LH35. Anti-CTLA-4 antibodies may contain one of the following combinations of light and heavy chains.

[0109] Table 6. Complete structures of ipilimumab and its variants

[0110]

[0111] In one example, the anti-CTLA-4 antibody exhibits reduced binding to CTLA-4 at approximately pH 5.5, and the anti-CTLA-4 antibody may be LH13, LH14, or LH25. In another example, the anti-CTLA-4 antibody exhibits reduced binding to CTLA-4 at approximately pH 6.0, and the anti-CTLA-4 antibody may be LH25.

[0112] LH25 includes a light chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 1, CDR2 containing the sequence shown in SEQ ID NO: 3, and CDR3 containing the sequence shown in SEQ ID NO: 5. LH25 also includes a heavy chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 7, CDR2 containing the sequence shown in SEQ ID NO: 8, and CDR3 containing the sequence shown in SEQ ID NO: 12. LH25 includes a light chain variable region containing the sequence shown in SEQ ID NO: 16 and a heavy chain variable region containing the sequence shown in SEQ ID NO: 28. LH25 includes a light chain containing the sequence shown in SEQ ID NO: 21 and a heavy chain containing the sequence shown in SEQ ID NO: 35.

[0113] LH13 includes a light chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 2, CDR2 containing the sequence shown in SEQ ID NO: 3, and CDR3 containing the sequence shown in SEQ ID NO: 4. LH13 also includes a heavy chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 6, CDR2 containing the sequence shown in SEQ ID NO: 10, and CDR3 containing the sequence shown in SEQ ID NO: 12. LH13 includes a light chain variable region containing the sequence shown in SEQ ID NO: 15 and a heavy chain variable region containing the sequence shown in SEQ ID NO: 26. LH13 also includes a light chain containing the sequence shown in SEQ ID NO: 20 and a heavy chain containing the sequence shown in SEQ ID NO: 33.

[0114] LH14 includes a light chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 2, CDR2 containing the sequence shown in SEQ ID NO: 3, and CDR3 containing the sequence shown in SEQ ID NO: 4. LH14 also includes a heavy chain variable region comprising CDR1 containing the sequence shown in SEQ ID NO: 6, CDR2 containing the sequence shown in SEQ ID NO: 11, and CDR3 containing the sequence shown in SEQ ID NO: 12. LH14 includes a light chain variable region containing the sequence shown in SEQ ID NO: 15 and a heavy chain variable region containing the sequence shown in SEQ ID NO: 27. LH14 also includes a light chain containing the sequence shown in SEQ ID NO: 20 and a heavy chain containing the sequence shown in SEQ ID NO: 34.

[0115] 3. Pharmaceutical Composition

[0116] This document provides compositions comprising anti-CTLA-4 antibodies. The compositions may contain pharmaceutically acceptable excipients. The compositions may contain sterile solutions for intravenous infusion. The compositions may be preservative-free aqueous solutions and may be suitable for parenteral administration. In one example, the composition independently contains an amount of anti-CTLA-4 antibody of about 5, 10, 20, 30, 40, or 50 mg / mL, or within a range of two of these amounts. The composition may contain about 5, 10, or 20 mg / mL of anti-CTLA-4 antibody. In one example, the composition contains 10 mg / mL of anti-CTLA-4 antibody. In one example, the composition contains 100 mg of anti-CTLA-4 antibody. The composition may contain about 5, 10, 15, 20, 25, or 30 mL of an aqueous solution of antibody. The aqueous solution may be contained in a vial. The volume of the aqueous solution in the vial may be 10 mL. In one instance, the vial was filled with a 10 mL extractable volume, totaling approximately 100 mg of anti-CTLA-4 antibody per vial.

[0117] Pharmaceutically acceptable carriers may comprise one or more of histidine buffers, acetate buffers, trehalose, PS80, sucrose, and EDTA. The composition may comprise about 5, 10, 15, 20, 25, 30, 35, or 40 mM histidine buffer, or amounts within a range of two of these amounts. In one example, the composition comprises 20 mM histidine buffer. The composition may also contain about 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, or 10.0% (w / v) α,α-trehalose dihydrate, or amounts within a range of two of these amounts. In one example, the composition contains 8.8% (w / v) α,α-trehalose dihydrate. The composition may contain about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 (w / v) of PS80, or an amount within a range of two of these amounts. In one example, the composition contains 0.06% (w / v) PS80. The composition may contain about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, or 0.40 mM EDTA, which may be EDTA•2Na•2H2O. In one example, the composition contains 0.2 mM EDTA. The composition may have a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or a pH within a range thereof.

[0118] Anti-CTLA-4 antibodies can be administered and co-formulated with a second cancer therapy. The second cancer therapy can be an anti-PD-1 antibody, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or the antibody described in US Patent No. 11,345,754, the contents of which are incorporated herein by reference. Anti-PD-1 antibodies can comprise a light chain containing the sequence shown in SEQ ID NO: 36 and a heavy chain containing the sequence shown in SEQ ID NO: 37, as described below.

[0119] Light chain

[0120] DIQLTQSPSFLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 36)

[0121] Heavy chain

[0122] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMSWVRQAPGKGLEWVSTISGGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPYGNYGMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 37)

[0123] In one example, the composition independently comprises about 5, 10, 20, 30, 40, or 50 mg / mL, or amounts within two of these ranges, of each of the anti-CTLA-4 antibody and the anti-PD-1 antibody. The composition may comprise about 5, 10, or 20 mg / mL of each of the anti-CTLA-4 antibody and the anti-PD-1 antibody. The composition may comprise an anti-CTLA-4 antibody to anti-PD-1 antibody ratio of about 10:1, 5:1, 4:1, 3:1, 2:1, 4:3, 3:2, 1:1, 2:3, 3:4, 1:2, 1:3, 1:4, 1:5, or 1:10. In one example, the composition comprises 10 mg / mL of each of the anti-CTLA-4 antibody and the anti-PD-1 antibody. In one example, the composition comprises 100 mg of each of the anti-CTLA-4 antibody and the anti-PD-1 antibody. The composition may contain about 5, 10, 15, 20, 25, or 30 mL of aqueous antibody solution. The aqueous solution may be contained in a vial. The volume of the aqueous solution in the vial may be 10 mL. In one example, anti-CTLA-4 and anti-PD-1 antibodies are present at a combined protein concentration of 20 mg / mL. Each vial may be filled with a 10 mL extractable volume, totaling approximately 100 mg / vial of anti-CTLA-4 antibody and 100 mg / vial of anti-PD-1 antibody.

[0124] 4. Dosing regimen

[0125] Anti-CTLA-4 antibodies can be administered systemically, either by injection or intravenously (IV). One or more doses of anti-CTLA-4 antibodies may be administered to a subject or intended for administration to a subject. Independently, each dose of anti-CTLA-4 antibody may be an amount of about 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg, or a range of two of these amounts. For subjects weighing 30 kg or more, each dose of anti-CTLA-4 antibody may be an amount of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg, or a range of two of these amounts. For subjects weighing 30 kg or more, each dose of anti-CTLA-4 antibody may be approximately 50-1000 mg.

[0126] When the dose of anti-CTLA-4 antibody is 100 mg, it may be infused over a period of at least 30 minutes or is intended to be infused over a period of at least 30 minutes. When the dose of anti-CTLA-4 antibody is 200 mg or more, it may be infused over a period of at least 60 minutes or is intended to be infused over a period of at least 60 minutes.

[0127] The dose of anti-CTLA-4 antibody can be approximately 1, 2, 3, 6, 10, 15, or 20 mg / kg, or a range of two of these amounts. A dose may contain 1–20 mg / kg of anti-CTLA-4 antibody. A dose may be 50–1000 mg of anti-CTLA-4 antibody. The subject's weight may be at least 30 kg.

[0128] Anti-CTLA-4 antibodies can be administered periodically, with each administration of one of the aforementioned doses to the subject. The dose may differ from the previous dose in each dosing cycle. Dosing may involve gradually increasing the dose. In one example, anti-CTLA-4 antibodies are administered approximately every 1, 2, 3, 4, 5, or 6 weeks. Specifically, anti-CTLA-4 antibodies are administered approximately every 3 weeks. When describing the duration of a dosing cycle, "approximately" may mean ±1, 2, or 3 days.

[0129] In one instance, the dose of the anti-CTLA-4 antibody is 10 mg / kg. The anti-CTLA-4 antibody can be administered using a dosing regimen comprising two doses of 10 mg / kg followed by extended dosing of 1–6 mg / kg (i.e., each subsequent dose is 1–6 mg / kg). Extended dosing may include administration of doses of 3 mg / kg or 6 mg / kg. In one instance, each administration is given approximately every 3 weeks. In another instance, the anti-CTLA-4 antibody is administered approximately every 4 weeks. Dosing may occur within or at a timeframe of approximately 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, or 52 weeks.

[0130] Anti-CTLA-4 antibodies can be administered in flat doses, which may contain approximately 200 mg (approximately 1.5 mg / kg per dose), 400 mg (approximately 3 mg / kg per dose), 600 mg (approximately 4.5 mg / kg per dose), 800 mg (approximately 6.0 mg / kg per dose), or 1000 mg (approximately 7.5 mg / kg per dose), or a range of two of these amounts.

[0131] 5. Treatment methods

[0132] The antibody compositions described herein can be used to upregulate immune responses. Upregulation of the immune system is particularly desirable in the treatment of cancer and chronic infections, and therefore the antibody compositions described herein are effective in treating such conditions. As used herein, the term "cancer" refers to a growth or tumor caused by the abnormal, uncontrolled growth of cells. "Cancer" explicitly includes leukemia and lymphoma. The term "cancer" also refers to a disease involving cells with the potential to metastasize to distant sites.

[0133] The antibody compositions described herein can be used to prepare pharmaceuticals. The compositions can also be administered to subjects requiring treatment. Subjects can be human. Subjects may require treatment for the diseases or conditions described herein.

[0134] Cancer can be carcinoma, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck squamous cell carcinoma (HNSCC), endometrial cancer, renal cell carcinoma, thyroid cancer, and skin cancer; including squamous cell carcinoma; lymphoid hematopoietic malignancies, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Berketts lymphoma; and myeloid hematopoietic malignancies, including acute and chronic myeloid leukemia and promyelocytic leukemia. Leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma. Cancers arising from apoptosis are also anticipated to be treatable by the methods and compositions of this invention. Such cancers may include, but are not limited to, follicular lymphoma, cancers with p53 mutations, hormone-dependent tumors of the breast, hormone-dependent tumors of the prostate and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In certain embodiments, the methods and compositions disclosed herein are used to treat or prevent malignant tumors or dysproliferative changes (e.g., metaplasia and developmental abnormalities) or hyperproliferative disorders, including those of the ovaries, bladder, esophagus, gastrointestinal region, liver, bile ducts, adenoids, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, the methods and compositions of the present invention are used to treat or prevent sarcomas, melanomas, or leukemia.

[0135] In one instance, the cancer is melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), or triple-negative breast cancer (TNBC). In a further instance, NSCLC is PD-1 resistant. Ovarian cancer can be high-grade serous ovarian cancer, which can be primary peritoneal cancer or fallopian tube cancer. TNBC can be PD-1 resistant. Melanoma can be immuno-oncology (IO) resistant.

[0136] In one instance, the cancer is a solid tumor. A solid tumor can be one or more of the following: histologically confirmed and cytologically confirmed. The tumor can be in a subject with progressive locally advanced or metastatic disease. The subject may demonstrate failure or intolerance to at least one established standard medical anticancer therapy. The subject's cancer diagnosis can be determined by standards of practice, such as the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology.

[0137] The antibody composition and its antigen-binding fragment can be used in conjunction with another antitumor therapy, which may be selected from, but is not limited to, current standard and experimental chemotherapy, hormone therapy, biotherapy, immunotherapy, radiotherapy, and surgery. In some embodiments, the compositions described herein may be administered in combination with therapeutic or preventative amounts of one or more agents, therapeutic antibodies, or other agents known to those skilled in the art for the treatment or prevention of cancer, autoimmune diseases, infectious diseases, or poisoning. Such agents include, for example, any of the above-described biological response modulators, cytotoxins, antimetabolites, alkylating agents, antibiotics, antimitotic agents, or immunotherapeutic agents.

[0138] Antibody compositions and their antigen-binding fragments can be used in conjunction with another antitumor immunotherapy. In such embodiments, the composition is administered in combination with molecules that disrupt or enhance the activity of alternative immunomodulatory pathways (e.g., PD-1, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27, or LAG3) or regulatory effector molecules such as cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-β, IFNg, Flt3, BLys) and chemokines (e.g., CCL21) to enhance immunomodulatory effects. Specific embodiments include bispecific antibodies comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof described herein in combination with anti-PD-1, anti-B7-H1 (atezolizumab (Tecentriq) or durvalumab (Imfinzi)), anti-B7-H3, anti-B7-H4, anti-LIGHT, anti-LAG3, anti-TIM3, anti-TIM4, anti-CD40, anti-OX40, anti-GITR, anti-BTLA, anti-CD27, anti-ICOS, or anti-4-1BB. In yet another embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered in combination with molecules (such as IDO inhibitors) that activate different phases or aspects of the immune response to achieve a broader immune response.

[0139] 6. Generate

[0140] The anti-CTLA-4 antibody and its antigen-binding fragment described herein can be prepared using a eukaryotic expression system. The expression system may require expression from a vector in mammalian cells such as Chinese hamster ovary (CHO) cells. The antibody can also be generated from a stable cell line that expresses the antibody from a vector or a portion of a vector already integrated into the cell's genome.

[0141] The anti-CTLA-4 antibody and its antigen-binding fragment described herein can be purified using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, the fusion protein can be engineered to contain an additional domain containing an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, the antibody described herein containing the Fc region of an immunoglobulin domain can be isolated from cell culture supernatant or cytoplasmic extract using a protein A or protein G column. Furthermore, tags such as c-myc, hemagglutinin, polyhistidine, or flags can be used. TMKodak tags can be used to aid in antibody purification. These tags can be inserted anywhere within the peptide sequence, including the carboxyl or amino terminus. Other potentially useful fusions include enzymes that facilitate peptide detection, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify peptides.

[0142] The present invention has several aspects, which are illustrated by the following non-limiting embodiments.

[0143] Example 1

[0144] Ipilimumab variants exhibit reduced CTLA-4 binding activity at low pH

[0145] This example demonstrates an ipilimumab variant with reduced CTLA-4 binding activity at low pH. Based on previous research, anti-CTLA-4 antibodies that bind tightly within the intracellular compartment and cause lysosomal degradation of CTLA-4 can lead to persistent loss of surface CTLA-4, exacerbating intracellular adverse events (irAEs). This type of antibody is called a “pH-insensitive antibody” (pHIA) because the low pH found in the intracellular compartment maintains binding affinity for CTLA-4 and allows the antigen-antibody complex to be delivered to the lysosome for degradation. Data show that the clinical antibody ipilimumab is a pHIA, which explains its tendency to induce irAEs. Furthermore, by preferentially downregulating CTLA-4, ipilimumab is less effective in triggering ADCC, intratumoral regulatory T cell depletion, and rejection of large established tumors.

[0146] The inventors' objective was to reduce irreversible adverse events (irAEs) and improve the therapeutic efficacy of ipilimumab by altering the pH sensitivity of its binding to CTLA-4. Based on their previous work, they had learned to generate pH-sensitive variants by replacing tyrosine (Y) with histidine (H) in the complementarity-determining region (CDR) of the antibody. The tyrosine side chain does not ionize under physiological conditions, but the imidazole side chain in histidine has a pKa of approximately 6.0. Therefore, below the characteristic pH of endocytic vesicles (pH 6), the imidazole ring is largely protonated, presumably altering the CDR binding properties. pH-sensitive variants of ipilimumab were screened by applying a similar amino acid substitution strategy. Different combinations of Y-to-H mutations were designed against all CDR Y residues in ipilimumab to generate a total of 23 ipilimumab variants. The structures of these variants are as follows: Figure 1 As shown in the figure, and described in Table 6.

[0147] The pH sensitivity of the variant antibody to CTLA-4 binding compared to the natural ipilimumab (LH00) was investigated by ELISA (Figure 2). The first group of variants was generated by introducing Y to H substitutions only in the heavy chain CDR. Figure 2ApH-sensitive anti-CTLA-4 antibody (pHSA) HL12 was used as a standard. Clinical ipilimumab and investigational ipilimumab (LH00, generated with variants) were used as pH-insensitive anti-CTLA-4 antibody controls. Five variants (LH01-05) showed comparable binding capacity to ipilimumab at neutral pH and did not achieve significant improvement in pH-sensitive binding to CTLA-4 under acidic pH conditions. Figure 2A To improve this, the two Y atoms in the light chain CDR of these variants were also mutated to H (LH30-35), which resulted in a significant increase in pH sensitivity; however, these variants now significantly lose their binding affinity for CTLA-4 at neutral pH. Figure 2B These data indicate that at least one Y mutation in the light chain CDR is essential for ligand binding affinity of ipilimumab at neutral pH. To balance the effects of pH sensitivity and binding affinity under neutral conditions, a single Y-to-H mutation in the ipilimumab light chain CDR was combined with different Y-to-H mutations in the heavy chain CDR. Figure 2C and 2D Certain variants (LH10-12, LH15, and LH24) maintained comparable binding affinity to wild-type or clinical ipilimumab at neutral pH, but did not achieve a significant improvement in pH sensitivity binding to CTLA-4 under acidic pH conditions. Other variants, including LH13, LH14, and LH25, significantly increased pH sensitivity without reducing CTLA-4 binding at neutral pH. Figure 2C and 2D (Highlighted with a red box).

[0148] Head-to-head comparisons of these three variants with ipilimumab on the same plate using ELISA confirmed their improved pH sensitivity. Data showed that below pH 5.5 (characteristic of late endosomes and lysosomes), all three variants exhibited significantly reduced binding to CTLA-4. However, at approximately pH 6.0 and above (found in early endosomes), variants LH13 and LH14 maintained binding capacity, while LH25 dissociated from CTLA-4 (Figure 3). Therefore, it is concluded that LH25 is a pH 6.0 sensitive variant, while LH13 and LH14 are not. Further analysis of the properties of these two types of variants will follow.

[0149] Example 2

[0150] The pH-sensitive ipilimumab variant dissociates from CTLA-4 after endocytosis and does not reduce CTLA-4 on the cell surface.

[0151] This example demonstrates an ipilimumab variant exhibiting reduced binding to CTLA-4 at acidic pH. To determine whether the pH sensitivity of the ipilimumab variant leads to its dissociation from CTLA-4 in living cells, the antibody was incubated with 293T cells stably expressing human CTLA-4 at 4°C for 30 minutes. After washing away unbound antibody, the cells were switched to 37°C for 1 hour. After lysis with cell lysis buffer, the anti-CTLA-4 antibody was pulled down using protein G beads. Figure 4 As shown in the top figure, wild-type and other variants bound a considerable amount of CTLA-4. After incubation at 37°C, significantly less CTLA-4 associated with variant LH25 compared to LH00 (wild-type ipilimumab) or variants LH13 and LH14 (which are less sensitive (but not entirely insensitive) to dissociation at pH 6.0). Figure 4 Since both LH13 and LH14 are not very sensitive to binding at pH 6.0 (referred to in this paper as the "pH 6.0 insensitive variant"), LH13 was chosen as the prototype of the pH 6.0 insensitive antibody for further research (although it does exhibit pH sensitivity).

[0152] The binding affinity of these variants to CTLA-4 was compared by surface plasmon resonance (SPR) at pH 6.0 and pH 7.4. At pH 7.4, although both LH13 and LH25 showed slightly reduced affinity for CTLA-4 compared to ipilimumab, all antibodies showed comparable binding affinity to CTLA-4. Figure 5 (See the figure above). However, when the pH was reduced to 6.0, LH25 exhibited decreased affinity and a significantly faster dissociation rate compared to LH13, suggesting that LH25 may allow CTLA-4 recycling rather than lysosomal degradation. Figure 5 (See the image below). Figure 4 and 5 The data suggest that when the antibody-CTLA-4 complex is internalized into the endosome within the cell, the pH-6.0 sensitive variant is expected to dissociate from CTLA-4 and will be more readily released to be transported back to the plasma membrane, allowing for normal surface expression and immunomodulatory activity.

[0153] To confirm this finding, CTLA-4 levels in plasma membrane association after treatment with ipilimumab or its variants were evaluated using immunoblotting at the cellular level. Figure 6As shown, wild-type ipilimumab and its pH-6.0 insensitive variant LH13 induced a significant reduction in CTLA-4. Notably, the pH-6.0 sensitive variant LH25 allowed ipilimumab-induced downregulation of CTLA-4, but subsequently allowed for the recovery of surface CTLA-4. These data are consistent with the hypothesis that pH sensitivity at pH 6 is a good indicator of the longevity of antibody-mediated CTLA-4 reduction.

[0154] Example 3

[0155] pH-sensitive ipilimumab variants rescue CTLA-4 from lysosomal degradation

[0156] Previous studies have shown that pH-insensitive target binding of antibodies triggers lysosomal degradation of CTLA-4, which is associated with a lack of surface CTLA-4 and irAE. To test whether an ipilimumab variant with improved pH sensitivity could rescue CTLA-4 from lysosomal degradation, anti-CTLA-4 antibody was labeled with AF488 and incubated with CTLA-4-expressing CHO cells at 4°C, followed by washing away unbound antibody. Figure 7 As shown in the left image, all antibodies uniformly labeled CTLA-4 on the cell surface (green). After switching CHO cells to 37°C, all anti-CTLA-4 antibodies were also internalized. However, the internalized antibodies exhibited different destinations within the cells. Both the wild-type ipilimumab bound to the cell surface and the pH-6.0 insensitive variant LH13 co-localized with the lysotracker, indicating lysosomal deposition of the antibodies. Figure 7 (See right figure). In contrast, the pH-6.0 sensitive variant LH25 does not localize to lysosomes ( Figure 7 (See right image).

[0157] Because CHO cells express CTLA-4 with an orange fluorescent protein (OFP) tag, it is possible to simultaneously track the endpoints of antibodies and CTLA-4 within the cell. Figure 8 As shown, both wild-type ipilimumab and LH13 co-localize with CTLA-4 and lysotracker (white arrows), indicating that they both persistently associate with CTLA-4 and may promote CTLA-4 targeting to lysosomes. In contrast, most LH25-containing vesicles lack CTLA-4, suggesting that LH25 dissociates from CTLA-4 after endocytosis to avoid lysosomal deposition. These data indicate that while the pH-6.0 insensitive ipilimumab variants maintain binding to CTLA-4 and significantly drive CTLA-4 entry into lysosomes, the pH-6.0 sensitive variants dissociate from CTLA-4 after endocytosis and evade lysosomal targeting.

[0158] Example 4

[0159] The pH 6.0 sensitive ipilimumab variant was less toxic than ipilimumab.

[0160] To evaluate the effect of pH sensitivity on antibody-induced irAEs, young Ctla4 cells that had also received anti-PD-1 therapy to sensitize them to irAEs were studied. h / h In mice (humanized CTLA-4 knock-in mice), ipilimumab, the pH-6.0 insensitive variant LH13, and the pH-6.0 sensitive variant LH25 were compared. Mice were treated at days 10, 13, 16, and 19 postnatal, and body weight gain over time, hematological changes, and histopathological changes were evaluated 30 days after treatment (Figures 9 and 10). Figure 9A As shown, although mice showed substantial and statistically significant growth retardation in response to anti-PD-1 + ipilimumab or anti-PD-1 + LH13, no growth retardation was observed when mice received anti-PD-1 + LH25.

[0161] To investigate the effect of pH sensitivity on anemia, a total blood cell count was performed one month after the initiation of combination therapy. Figure 9B As expected, significant reductions in hematocrit (HCT), total hemoglobin (Hb), and mean corpuscular volume (MCV) were observed in most mice treated with ipilimumab + anti-PD-1 or LH13 + anti-PD-1, while mice receiving LH25 + anti-PD-1 did not show such effects. Figure 9B In the LH25 + anti-PD-1 group, red blood cell counts (RBCs) were largely normal, while in both the ipilimumab + anti-PD-1 and LH13 + anti-PD-1 groups, RBC counts were decreased, although the decrease in the LH13 + anti-PD-1 group was not statistically significant. Figure 9B These data indicate that pH-6.0 sensitive variants, rather than pH-6.0 insensitive variants, do not cause the anemia observed with ipilimumab plus anti-PD1 combination therapy.

[0162] To quantitatively analyze the effects of ipilimumab variants on tissue damage, histological analysis was performed on viscera and glands from the experimental mice shown in Figure 9. Organs and glands (lung, heart, liver, and salivary glands) were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin (H & E), and scored using a double-blind method. Representative tissue sections are shown in Figure 9. Figure 10A In the middle; the scores from individual mice in each group are presented in Figure 10BAs shown in Figure 10, when combined with anti-PD-1, wild-type ipilimumab and its variant LH13 induced inflammation in all mice, with severe inflammation observed in all major organs. Notably, inflammation was largely eliminated in mice receiving variant LH25 plus anti-PD-1 treatment. Based on the scores for each organ, it is clear that ipilimumab or LH13 + anti-PD-1 induced significantly more severe inflammation than LH25 + anti-PD-1 treatment. Figure 10B In summary, these studies demonstrate that treatment with a pH-6.0 sensitive variant, rather than a pH-6.0 insensitive variant, alleviates ipilimumab-induced irAE.

[0163] Example 5

[0164] Increased intratumor Treg reduction and antitumor activity of pH-6 sensitive ipilimumab variants

[0165] This example demonstrates that, compared to ipilimumab, the pH-sensitive ipilimumab variant increases intratumoral Treg reduction and antitumor activity. To test the effectiveness of the pH-sensitive ipilimumab variant in inducing tumor rejection, Ctla4 was subcutaneously inoculated using two different cancer cell models. h / h Mice (humanized CTLA-4 knock-in mice): YM3.3 melanoma and MC38 colon cancer cells. In the YM3.3 melanoma model, mice were treated with three doses of ipilimumab (1.5 mg / kg / dose), LH13, LH25, or IgG control on days 10, 13, and 16 post-tumor inoculation. The same treatment was performed in the MC38 colon cancer model on days 17 and 20 post-tumor inoculation with two doses of antibody. Tumor growth was observed by measuring tumor size every three days. Figure 11A As shown, at these limiting doses, all antibodies induced a slowing of tumor growth in both models.

[0166] Although the reduction in tumor growth in the LH13 group was not significantly different from that in the IgG control group, LH25 was significantly more effective in shrinking tumors compared to wild-type ipilimumab and LH13. Survival curves of tumor-bearing mice treated with different antibodies were further analyzed using the criterion of "death" when the tumor diameter reached 2 cm. Figure 11B As shown, in both melanoma and colon cancer models, although LH13 exhibited the same survival rate as wild-type ipilimumab, mice receiving LH25 had significantly longer survival times.

[0167] It has been demonstrated that the selective reduction of tumor Tregs by anti-CTLA-4 antibodies is key to their in vivo anti-tumor effect. Therefore, the effect of a pH-sensitive ipilimumab variant on the reduction of intratumoral Tregs was evaluated. The antibody was injected into mice challenged with YM3.3 tumors. On day 14, the pH-sensitive variant LH25 significantly reduced intratumoral Tregs but did not reduce Tregs from the spleen. Figure 12 It is evident that wild-type ipilimumab and LH13 are less effective than LH25 in tumor Treg reduction. These data reveal that pH-6.0 sensitive variants, rather than pH-6.0 insensitive variants, can improve the therapeutic effect of ipilimumab by causing substantial Treg reduction.

[0168] In summary, the data presented herein demonstrate that antibodies that maintain binding to CTLA-4 at an acidic pH of 6.0 contribute to irAEs (irAEs). This is because pH-6.0 insensitive antibody variants maintain CTLA-4 binding, leading to lysosomal targeted degradation of CTLA-4. Clearly, the resulting loss of CTLA-4 surface expression promotes an unregulated immune response leading to irAEs. In contrast, pH-6.0 sensitive ipilimumab promotes CTLA-4 recycling to the cell surface and prevents its lysosomal degradation, thereby reducing the tendency for irAEs. Furthermore, the pH-6.0 sensitive variants described herein also trigger better antitumor efficacy by more effectively attenuating Tregs in tumors than other antibodies tested. In conclusion, these examples demonstrate that pH 6.0 sensitivity of anti-human CTLA-4 antibodies is associated with improved immunotherapeutic efficacy and reduced adverse inflammatory effects.

Claims

1. An anti-CTLA-4 antibody comprising: (a) a light chain variable region comprising a complementarity determining region (CDR) 1 comprising the sequence set forth in SEQ ID NO: 1, a CDR2 comprising the sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the sequence set forth in SEQ ID NO: 5; and a heavy chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 7, a CDR2 comprising the sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the sequence set forth in SEQ ID NO: 12; (b) a light chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 2, a CDR2 comprising the sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the sequence set forth in SEQ ID NO: 4; and a heavy chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the sequence set forth in SEQ ID NO: 12; or (c) a light chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 2, a CDR2 comprising the sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the sequence set forth in SEQ ID NO: 4; and a heavy chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the sequence set forth in SEQ ID NO:

12.

2. The anti-CTLA-4 antibody of claim 1, wherein the light chain variable region comprises the complementarity determining region (CDR) 1 comprising the sequence set forth in SEQ ID NO: 1, the CDR2 comprising the sequence set forth in SEQ ID NO: 3, and the CDR3 comprising the sequence set forth in SEQ ID NO: 5; and the heavy chain variable region comprises the CDR1 comprising the sequence set forth in SEQ ID NO: 7, the CDR2 comprising the sequence set forth in SEQ ID NO: 8, and the CDR3 comprising the sequence set forth in SEQ ID NO:

12.

3. The anti-CTLA-4 antibody of claim 2, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 16, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO:

28.

4. The anti-CTLA-4 antibody of claim 3, comprising a light chain comprising the sequence set forth in SEQ ID NO: 21 and a heavy chain comprising the sequence set forth in SEQ ID NO:

35.

5. The anti-CTLA-4 antibody of claim 1, wherein the light chain variable region comprises the CDR1 comprising the sequence set forth in SEQ ID NO: 2, the CDR2 comprising the sequence set forth in SEQ ID NO: 3, and the CDR3 comprising the sequence set forth in SEQ ID NO: 4; and the heavy chain variable region comprises the CDR1 comprising the sequence set forth in SEQ ID NO: 6, the CDR2 comprising the sequence set forth in SEQ ID NO: 10, and the CDR3 comprising the sequence set forth in SEQ ID NO:

12.

6. The anti-CTLA-4 antibody of claim 5, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 15, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO:

26.

7. The anti-CTLA-4 antibody of claim 6, comprising a light chain comprising the sequence set forth in SEQ ID NO: 20 and a heavy chain comprising the sequence set forth in SEQ ID NO:

33.

8. The anti-CTLA-4 antibody of claim 1, wherein the light chain variable region comprises the CDR1 comprising the sequence set forth in SEQ ID NO: 2, the CDR2 comprising the sequence set forth in SEQ ID NO: 3, and the CDR3 comprising the sequence set forth in SEQ ID NO: 4; and the heavy chain variable region comprises the CDR1 comprising the sequence set forth in SEQ ID NO: 6, the CDR2 comprising the sequence set forth in SEQ ID NO: 11, and the CDR3 comprising the sequence set forth in SEQ ID NO:

12.

9. The anti-CTLA-4 antibody of claim 5, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 15, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO:

27.

10. The anti-CTLA-4 antibody of claim 6, comprising a light chain comprising the sequence set forth in SEQ ID NO: 20 and a heavy chain comprising the sequence set forth in SEQ ID NO:

34.

11. The anti-CTLA-4 antibody of claim 1, wherein the anti-CTLA-4 antibody binds to CTLA-4 with reduced affinity at a pH of about 5.5-6.0 compared to ipilimumab.

12. The anti-CTLA-4 antibody of claim 2, wherein the anti-CTLA-4 antibody binds to CTLA-4 with reduced affinity at a pH of about 6.0 compared to ipilimumab.

13. A composition comprising the anti-CTLA-4 antibody of claim 1 and a pharmaceutically acceptable excipient.

14. The composition of claim 13, comprising 20 mM histidine buffer, 8.8% (w / v) a,a-trehalose dihydrate, 0.06% (w / v) PS80, and 0.2 mM EDTA 2Na 2H20, wherein the composition has a pH of about 6.

0.

15. The composition of claim 13, further comprising an anti-PD-1 antibody.

16. The composition of claim 15, wherein the anti-PD-1 antibody comprises a light chain comprising the sequence set forth in SEQ ID NO: 36 and a heavy chain comprising the sequence set forth in SEQ ID NO:

37.

17. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-CTLA-4 antibody of claim 1.

18. The method of claim 17, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular cancer, cholangiocarcinoma, adenoid cystic carcinoma (ACC), and triple negative breast cancer (TNBC).

19. The method of claim 17, wherein the cancer is a solid tumor.

20. The method of claim 17, wherein the anti-CTLA-4 antibody or the composition is administered intravenously.

21. The method of claim 17, comprising administering to the subject up to 10 mg / kg of the anti-CTLA-4 antibody.

22. The method of claim 21, wherein the administration is once every 3 weeks.

23. Use of an anti-CTLA-4 antibody of any one of claims 1-12 in the manufacture of a medicament for treating cancer.

24. The use of claim 23, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular cancer, cholangiocarcinoma, adenoid cystic carcinoma (ACC), and triple negative breast cancer (TNBC).

25. The use of claim 23, wherein the cancer is a solid tumor.

26. The use of claim 23, wherein the anti-CTLA-4 antibody is for intravenous administration.

27. The use of claim 23, wherein up to 10 mg / kg of the anti-CTLA-4 antibody is to be administered.

28. The use of claim 27, wherein the administration is once every 3 weeks.

29. An anti-CTLA-4 antibody of any one of claims 1-12 for use in treating cancer.

30. The anti-CTLA-4 antibody of claim 29, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular cancer, cholangiocarcinoma, adenoid cystic carcinoma (ACC), and triple negative breast cancer (TNBC).

31. The anti-CTLA-4 antibody of claim 29, wherein the cancer is a solid tumor.

32. The anti-CTLA-4 antibody of claim 29, wherein the anti-CTLA-4 antibody is for intravenous administration.

33. The anti-CTLA-4 antibody of claim 32, wherein 10 mg / kg of the anti-CTLA-4 antibody is to be administered.

34. The anti-CTLA-4 antibody of claim 33, wherein the administration is once every 3 weeks.

35. A pharmaceutical composition comprising the anti-CTLA-4 antibody of any one of claims 1-12 for use in treating cancer.

36. The pharmaceutical composition of claim 35, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular cancer, cholangiocarcinoma, adenoid cystic carcinoma (ACC), and triple negative breast cancer (TNBC).

37. The pharmaceutical composition of claim 35, wherein the cancer is a solid tumor.

38. The pharmaceutical composition of claim 35, wherein the anti-CTLA-4 antibody is for intravenous administration.

39. The pharmaceutical composition of claim 35, wherein 10 mg / kg of the anti-CTLA-4 antibody is to be administered.

40. The pharmaceutical composition of claim 39, wherein the administration is once every 3 weeks.

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