Chimeric and humanized anti-human ctla4 monoclonal antibodies and uses thereof

Abstract

The present invention relates to compositions of chimeric and humanized antibodies that bind to the human CTLA4 molecule and their use in cancer immunotherapy and for reducing autoimmune side effects compared to other immunotherapeutics.

Description

[0001] This application is a divisional application of patent application No. 201680073425.2, filed on December 14, 2016, with a priority date of December 15, 2015, entitled "Chimeric and Humanized Anti-Human CTLA4 Monoclonal Antibody and Its Use". Technical Field

[0002] This invention relates to chimeric and humanized antibodies that bind to human CTLA4 molecules and methods of using them. Background Technology

[0003] The immune system in humans and other mammals is responsible for providing protection against infection and disease. This protection is provided by both humoral and cell-mediated immune responses. Humoral responses result in the production of antibodies and other biomolecules that can recognize and neutralize foreign targets (antigens). In contrast, cell-mediated immune responses involve the activation of macrophages, neutrophils, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes by T cells, as well as the release of various cytokines in response to antigen recognition.

[0004] The ability of T cells to optimally mediate an immune response against an antigen requires two distinct signaling interactions. First, the antigen, lining the surface of antigen-presenting cells (APCs), must be presented to antigen-specific primary T cells in the form of an MHC:peptide complex (1,2). This presentation is signaled via T cell receptors (TCRs), which guide T cells to initiate a specific immune response to the presented antigen. Second, a series of co-stimulatory signals mediated by the interaction between APCs and unique T cell surface molecules first trigger T cell activation and proliferation and ultimately trigger their inhibition (3-5). Thus, the first signal confers specificity to the immune response, while the second signal determines the nature, magnitude, and duration of the response while confining the immunity to itself. Of particular importance among these second signaling molecules is the binding between the B7.1 (CD80) (6) and B7.2 (CD86) (7-9) ligands of antigen-presenting cells and the CD28 and CTLA4 receptors of T lymphocytes (10-12).

[0005] Cytotoxic T-lymphocyte antigen-4 (CTLA4) is considered a key regulator of adaptive immune responses, playing a crucial role in maintaining peripheral tolerance and shaping the emerging T-cell response repertoire, and is therefore a therapeutic target for cancer and inflammation. Treatment with anti-CTLA4 antibodies has shown to be a powerful tool for enhancing anti-tumor immunity in preclinical models (10). Monotherapy with antibodies targeting CTLA4 promotes rejection of transplantable tumors from various sources.

[0006] Based on promising preclinical tumor model studies, the clinical potential of antibodies targeting CTLA4 in various human malignancies has been explored. Although anti-CTLA4 (ipilimumab, marketed by Yervoy) has demonstrated efficacy in treating melanoma, CTLA4 treatment and targeting are associated with autoimmune toxicity. Characteristic side effects from CTLA4 inhibition are commonly referred to as immune-related adverse events (irAEs), and the most common irAEs are rash, hepatitis, colitis, and endocrine disorders, particularly hypopituitarism. Therefore, there is a need to improve the therapeutic potential of anti-CTLA4 antibodies by increasing efficacy while reducing associated irAEs.

[0007] Another focus in immunotherapy and cancer treatment is combining different immune checkpoint inhibitors to enhance anti-tumor activity, especially against poorly immunogenic tumors. However, this approach is associated with the risk of further increasing autoimmune side effects, further highlighting the need to selectively modulate cancer immunity without enhancing autoimmunity.

[0008] Further research on ligands for the CD28 receptor has identified and characterized a group of related B7 molecules (“B7 superfamily”) (32-33). Several family members are currently known: B7.1 (CD80), B7.2 (CD86), inducible costimulatory factor ligand (ICOS-L), programmed death-1 ligand (PD-L1; B7-H1), programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4, and B7-H6 (35-36).

[0009] B7-H1 is widely expressed in various human and mouse tissues, such as the heart, placenta, muscle, fetal liver, spleen, lymph nodes, and thymus in both species, as well as the liver, lungs, and kidneys in mice only (37). B7-H1 (PD-L1, CD274) is a particularly important member of the B7 superfamily because it plays a key role in shaping the immune response to tumors (38; U.S. Patent Nos. 6,803,192, 7,794,710; U.S. Patent Application Publications Nos. 2005 / 0059051, 2009 / 0055944, 2009 / 0274666, 2009 / 0313687; PCT Publications Nos. WO 01 / 39722, WO 02 / 086083).

[0010] Programmed death-1 (“PD-1”) is the receptor for B7-H1 and B7-DC. PD-1 is a type I membrane protein member of the CD28 / CTLA4 family of T cell regulatory factors (39; U.S. Patent Application Publications Nos. 2007 / 0202100, 2008 / 0311117, 2009 / 00110667; U.S. Patents Nos. 6,808,710, 7,101,550, 7,488,802, 7,635,757, 7,722,868; PCT Publication No. WO 01 / 14557). Compared to CTLA4, PD-1 more broadly and negatively regulates the immune response. PD-1 is expressed on activated T cells, B cells, and monocytes (40-41) and is expressed at low levels on natural killer (NK) T cells (42-43).

[0011] The interaction between B7-H1 and PD-1 has been found to provide key negative co-stimulatory signals to T and B cells (43) and act as a cell death inducing factor (39). The roles of B7-H1 and PD-1 in inhibiting T cell activation and proliferation have suggested that these biomolecules could serve as therapeutic targets for inflammation and cancer. Therefore, the use of anti-PD1 and anti-B7-H1 antibodies for treating infections and tumors, as well as upregulating adaptive immune responses, has been proposed and proven effective in treating a variety of human tumors. However, not all subjects respond to or have a complete response to anti-PD-1 or anti-B7-H1 treatment, and therefore there is considerable interest in combining anti-PD-1 or anti-B7-H1 antibodies with other immune checkpoint inhibitors to enhance antitumor activity.

[0012] 4-1BB (also known as CD137 and TNFRSF9) is another immune checkpoint molecule. The most characteristic activity of CD137 is its co-stimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Furthermore, similar to anti-CTLA4, anti-4-1BB antibodies can enhance immune activity in mice to eliminate tumors (27-29). However, unlike anti-CTLA4 antibodies, which tend to exacerbate autoimmune diseases, cancer-therapeutic anti-4-1BB mAbs have been shown to eliminate the occurrence of autoimmune diseases in mice susceptible to lupus, where they inhibit anti-dsDNA antibody production and alleviate renal pathology (25,26). Previous data have demonstrated the potential to reduce the autoimmune side effects of anti-CTLA4 therapy in a mouse model of colon cancer while enhancing antitumor activity by combining anti-CTLA4 therapy with anti-4-1BB antibodies (19). This suggests the potential to counteract the autoimmune side effects of anti-CTLA4 tumor therapy.

[0013] Preclinical screening of anti-human CTLA4 antibodies is fraught with challenges because in vitro immune relevance is sometimes of little value, as demonstrated by experience with anti-mouse CTLA4 antibodies. The same anti-mouse CTLA4 antibodies that induce potent antitumor immunity in vivo can exhibit variable effects on T cells in vitro. Anti-CTLA4 antibodies enhance T cell proliferation in response to allogeneic antigens but inhibit T cell proliferation in response to co-stimulation with anti-CD28 (30,31). Furthermore, CTLA4 binding to antibodies can promote or inhibit the proliferation of different T cell subsets in the same culture (32). This complexity could be overcome if human T cell responses in rodent models could be studied.

[0014] This article describes anti-CTLA4 antibodies used to enhance immune responses and reduce autoimmune side effects in antitumor therapy. Furthermore, these antibodies can be combined with other checkpoint inhibitors, such as anti-PD-1 and anti-4-1BB, to enhance antitumor activity while eliminating autoimmune side effects. Summary of the Invention

[0015] This invention relates to antibody compositions that bind to human CTLA4 molecules and their antigen-binding fragments, and their use in cancer immunotherapy and in reducing autoimmune side effects. Specifically, this invention relates to antibodies that enhance CTLA4 blocking activity, enhance effector function, or reduce binding to soluble CTLA4 relative to membrane-bound or immobilized CTLA4 by CTLA4 ligands B7.1 and B7.2.

[0016] The antibody may comprise a light chain variable amino acid sequence having an amino acid sequence comprising a light chain variable amino acid sequence having the amino acid sequence described in SEQ ID NO:1; and a heavy chain variable amino acid sequence having the amino acid sequence described in SEQ ID NO:2. The antibody may also comprise a heavy chain variable amino acid sequence having an amino acid sequence described in SEQ ID NO:27, 28, or 29; and a light chain variable amino acid sequence having an amino acid sequence described in SEQ ID NO:30, 31, or 32. The antibody may comprise a light chain variable region having a CDR sequence described in SEQ ID NO:21, 22, and 23; and a heavy chain variable region having a CDR sequence described in SEQ ID NO:24, 25, and 26. More specifically, the antibody may comprise a heavy chain variable region having a CDR2 sequence described in SEQ ID NO:33, 34, or 35; and a light chain variable region having a CDR sequence described in SEQ ID NO:36, 37, or 38.

[0017] The immunoglobulin heavy chain constant region of the antibody may contain the amino acid sequence described in SEQ ID NO:3 or 4. The immunoglobulin heavy chain constant region of the antibody may also contain mutations. The mutations relative to the hIgG1 backbone sequence in SEQ ID NO:3 may be M135Y, S137T, T139E, S181A, E216A, or K217A, or combinations thereof. Preferably, the immunoglobulin heavy chain constant region of the antibody may contain all six mutations. The antibody may contain a heavy chain amino acid sequence having the amino acid sequence described in SEQ ID NO:6; and a light chain amino acid sequence having the amino acid sequence described in SEQ ID NO:8. The antibody may also contain a heavy chain amino acid sequence having the amino acid sequence described in SEQ ID NO:9, 11, or 13; and a light chain amino acid sequence having the amino acid sequence described in SEQ ID NO:15, 17, or 19. The antibody is capable of binding to human CTLA4. The antibody may also inhibit the binding of human CTLA4 to B7-1 or B7-2.

[0018] This article further provides the antigen-binding fragments of the antibodies described herein.

[0019] This document also provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody described herein. The pharmaceutical composition may comprise a physiologically acceptable carrier or excipient.

[0020] In another aspect, methods for enhancing one or more immune functions or responses in a subject are presented herein, comprising administering the anti-CTLA4 antibody composition and pharmaceutical composition described herein to a subject in need. In one specific embodiment, methods for preventing, treating, and / or managing diseases requiring activation or enhancement of one or more immune functions or responses are presented herein. The disease may be cancer, which may be a human malignancy. Specifically, human malignancies may be melanoma, lung cancer, breast cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, Hodgkin's or non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, or renal cell carcinoma. In another embodiment, the disease to be treated is an infectious disease. The methods described herein can minimize autoimmune adverse effects associated with immunotherapy.

[0021] In other specific embodiments, the method comprises combination therapy, wherein a subject is given a combination of the anti-CTLA4 antibody composition described herein with another therapy that can activate or enhance one or more immune functions or responses. In another embodiment, the anti-CTLA4 antibody composition described herein is given in combination with an antigenic composition as an adjuvant. In one specific embodiment, the anti-CTLA4 antibody composition described herein is given in combination with a vaccine composition to induce or activate or enhance the immune response induced by the vaccine composition.

[0022] In one specific embodiment, the subject is given a combination of the anti-CTLA4 antibody composition described herein with one or more other therapies targeting different immune regulatory pathways. In a preferred embodiment, the activity of the therapies targeting different immune regulatory pathways is complementary or synergistic with the anti-CTLA4 antibody composition described herein. In one case, the anti-CTLA4 antibody composition described herein is given in combination with other checkpoint inhibitors or small tumor immunomodulators (such as indoleamine 2,3-dioxygenase (IDO) inhibitors). In another case, the anti-CTLA4 antibody composition described herein is given in combination with an immunostimulatory molecule. Specific embodiments include combining the anti-CTLA4 antibody composition described herein with anti-PD-1 (pembrolizumab (Keytruda) or nivolumab (Opdivo)), anti-B7-H1 (atezolizumab (Tecentriq) or durvalumab)), anti-B7-H3, anti-B7-H4, anti-LAG3, anti-Tim3, anti-CD40, anti-OX40, anti-BTLA, anti-CD27, anti-ICOS, or anti-41BB. In another embodiment, the anti-CTLA4 antibody composition described herein is combined with a second immunostimulatory molecule in a single bispecific antibody.

[0023] In another embodiment, the anti-human CTLA4 antibody described herein can preferentially bind to human CTLA-4 expressed on the cell surface relative to soluble CTLA4 molecules. The anti-human CTLA4 antibody can bind to human CTLA4 and preferentially upregulate the expression of B7.1 or B7.2 in vivo. The antibody can be contained in compositions for modulating immune responses (immunotherapy) and treating cancer.

[0024] This invention further relates to a method for screening anti-human CTLA4 mAbs with preferred activity. Preclinical screening of anti-human CTLA4 mAbs is fraught with difficulties because the in vitro immune correlation between cancer immunity and adverse autoimmune effects is uncertain. Significant autoimmune side effects have been observed in clinical trials of human anti-CTLA4 (ipilimumab), especially when combined with anti-PD-1. To identify anti-CTLA4 antibodies with reduced immune-related toxicity, the ability of antibodies exhibiting antitumor activity in humanized mice to reduce adverse autoimmune effects in vivo can be screened using human CTLA4 gene knock-in mice.

[0025] In another embodiment, the present invention relates to a method for screening anti-human CTLA4 mAb with enhanced anti-tumor effects, wherein the antibody exhibits enhanced local depletion of Treg cells in the tumor environment.

[0026] In another embodiment, the present invention relates to a method for monitoring the blocking effect of anti-CTLA4 antibodies in vivo by monitoring the expression levels of B7.1 and B7.2 on immune cells (such as antigen-presenting cells (APCs)). The invention further covers measuring the bioactivity of anti-CTLA4 antibodies in vivo and monitoring biomarkers of a clear response to anti-CTLA4 therapy by measuring the expression levels of B7.1 and B7.2 on in vitro immune cells.

[0027] To map the CTLA4 binding epitopes of the L3D10 parental antibody and its humanized variants PP4631 and PP4637, the fact that mouse and human CTLA4 proteins are cross-reactive with B7-1 but not with anti-CTLA-4 antibodies was utilized. Therefore, several mutants of the human CTLA-4 Fc protein were designed, in which amino acid clusters from the human CTLA-4 protein were replaced with amino acids from the mouse CTLA-4 protein. Because the anti-CTLA-4 antibody used in this study does not bind to mouse CTLA-4, the binding of the anti-human CTLA-4 antibody was eliminated when key residues of the antibody-binding epitope were replaced with mouse amino acids.

[0028] The present invention includes the following embodiments:

[0029] 1. An antibody comprising: (a) a light chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1; and (b) a heavy chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 2.

[0030] 2. An antibody comprising: (a) a heavy chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 27, 28 or 29; and (b) a light chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 30, 31 or 32.

[0031] 3. An antibody comprising: (a) a light chain variable region having the CDR sequences set forth in SEQ ID NO: 21, 22 and 23; and (b) a heavy chain variable region having the CDR sequences set forth in SEQ ID NO: 24, 25 and 26.

[0032] 4. The antibody according to any one of Examples 1 to 3, wherein the immunoglobulin heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 3 or 4.

[0033] 5. An antibody comprising: (a) a heavy chain amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 6; and (b) a light chain amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 8.

[0034] 6. An antibody comprising: (a) a heavy chain amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 9, 11 or 13; and (b) a light chain amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 15, 17 or 19.

[0035] 7. The antibody according to any one of Examples 1 to 6, wherein the antibody is capable of binding to human CTLA4.

[0036] 8. The antibody according to any one of Examples 1 to 7, wherein the antibody is characterized by reduced binding to soluble CTLA4.

[0037] 9. An antigen-binding fragment of an antibody according to any one of Examples 1 to 8.

[0038] 10. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 9 and a physiologically acceptable carrier or excipient.

[0039] 11. A method of treating cancer, comprising administering to a subject in need an effective amount of the pharmaceutical composition according to Example 10.

[0040] 12. The method according to Example 11, further comprising administering another agent selected from the group consisting of anti-PD-1 or anti-4-1BB.

[0041] 13. The method according to Example 12, wherein the anti-PD-1 or anti-4-1BB antibody and the anti-CTLA4 antibody are combined in a single molecule as bispecific antibodies.

[0042] 14. The method according to Example 11, wherein the pharmaceutical composition induces strong Treg loss and local T cell activation but minimal systemic T cell activation in the tumor microenvironment. Attached Figure Description

[0043] Figure 1 Schematic diagrams of chimeric (left) and humanized (right) L3D10 antibodies with novel combinations of mutations in the IgG1 Fc region. The location of the mutation in the Fc region is determined by its amino acid position number, and the amino acid is identified by its single-letter code. The letter before the number represents the substituted amino acid, and the letter after the number represents the introduced amino acid. The variable region of the antibody is depicted as a hollow ellipse, and the human sequence is depicted as a gray rectangle. V = variable region; C = constant region; L = light chain; H = heavy chain.

[0044] Figure 2 Chimeric L3D10 and 10D1 bound to plate-immobilized CTLA4 as determined by ELISA. ELISA plates were coated with 1 μg / ml CTLA4-His protein (Sino Biological, China). Predetermined concentrations of biotinylated binding protein were added, and binding was measured using HRP-bound streptavidin. 10D1-1 and -2 are two separate material batches of the same antibody. B7.1-Fc is a positive control, and Fc is a negative control.

[0045] Figure 3 L3D10 competition analysis. 10D1 is less efficient than chimeric L3D10 in blocking the binding of CTLA4. Experiments are as follows... Figure 2 The procedure was performed as described above, but the biotinylated chimeric L3D10 was mixed with a predetermined concentration of unlabeled CTLA4-binding protein or CTLA4-Fc before being added to the ELISA plate. It was noted that the blockade caused by unlabeled L3D10 was significantly better than that caused by 10D1, indicating that these antibodies have inconsistent binding sites.

[0046] Figure 4Blocking the binding of CTLA4 to plate-fixed B7.1. B7.1Fc protein was plated onto ELISA plates at a concentration of 0.5 μg / ml. After washing and blocking, biotinylated CTLA4-Fc protein was added at a concentration of 0.25 μg / ml in the presence of a competing protein. The data shown are the average of two densities at 405 nM. B7.1-Fc, chimeric L3D10, and CTLA4-Fc all blocked the CTLA4:B7.1 interaction in a dose-dependent manner, while two individual batches of the 10D1 antibody failed to block it at all tested doses. Biotinylation of CTLA4 did not destroy the 10D1 epitope on CTLA4, as both batches of 10D1 showed strong binding to biotinylated CTLA4 (data not shown).

[0047] Figure 5 Blocking the binding of CTLA4 to plate-fixed B7.2. B7.2 Fc protein was plated onto an ELISA plate at a concentration of 0.5 μg / ml. After washing and blocking, biotinylated CTLA4-Fc protein was added at a concentration of 0.25 μg / ml in the presence of a competing protein. Chimeric L3D10 blocked the CTLA4:B7.2 interaction in a dose-dependent manner, while two separate batches of the 10D1 antibody failed to completely block the CTLA4:B7.2 interaction even at the highest concentration.

[0048] Figure 6 Both .10D1 and L3D10 potently blocked the interaction between soluble B7-1 and B7-2 and B7-Fc-immobilized B7-CTLA4. Different doses of anti-human CTLA4 mAb were added to plates coated with human B7-1Fc along with 0.25 μg / ml biotinylated human CTLA4-Fc. The amount of CTLA4 bound to the plate was measured using HRP-bound streptavidin. The data shown are duplicated averages and represent two independent experiments.

[0049] Figure 7 Blocking the binding of CTLA4 to B7.1 expressed on the cell surface. Biotinylated CTLA4-Fc protein was added at 0.5 μg / ml to CHO cells expressing B7.1 in the presence of a predetermined concentration of competing protein. The binding of the biotinylated fusion protein to CHO cells transfected with mouse or human B7-1 and B7-2 was detected by flow cytometry. The amount of receptor bound was measured using phycoerythrin-streptolysin. The data shown are the mean fluorescence intensities of triplicate samples. Chimeric L3D10 blocked the CTLA4:B7.1 interaction in a dose-dependent manner, while two separate batches of the 10D1 antibody failed to block it at all tested doses.

[0050] Figure 8Blocking the binding of CTLA4 to mouse B7-1 expressed on the cell surface. When mB7-1 is expressed on CHO cells, 10D1 moderately but detectably blocked the mouse B7-1-human CTLA4 interaction. Different doses of anti-human CTLA4 mAb were added to CHO cells expressing mouse B7-1 along with 0.25 μg / ml human CTLA4-Fc. Data shown are mean and SEM or triplicate data representing two independent experiments.

[0051] Figure 9 Blocking the binding of CTLA4 to B7.2 expressed on the cell surface. Biotinylated CTLA4-Fc protein was added at 0.5 μg / ml to CHO cells expressing B7.2 in the presence of a competitive protein at a predetermined concentration. Chimeric L3D10 blocked the CTLA4:B7.2 interaction in a dose-dependent manner, while two separate batches of the 10D1 antibody failed to completely block the CTLA4:B7.2 interaction even at the highest concentration. The data shown in this figure have been replicated at least 5 times.

[0052] Figure 10 .10D1 binds to biotinylated human CTLA4-Fc better than L3D10. Different doses of anti-human CTLA4 mAb or control IgG were plated. Biotinylated CTLA4-Fc was added at 0.25 μg / ml. The amount of CTLA4 bound to the plate was measured using HRP-binding streptavidin. The data shown are the average of duplicates and represent two independent experiments.

[0053] Figure 11 L3D10, but not 10D1, blocked the interaction between multihistidine-labeled CTLA4 and CHO cells expressing human B7-1. CHO cells expressing human B7-1 were incubated with multihistidine-labeled CTLA4 and a predetermined dose of antibody, and the amount of CTLA4-Fc was detected using PE-streptin and measured by FACSCanto II. The data shown are average fluorescence intensities from triplicate samples and represent two independent experiments.

[0054] Figure 12 Complete remission of established tumors in a chimeric L3D10-induced syngeneic MC38 model. The top figure depicts the experimental design, and the bottom figure shows the growth kinetics of MC38 tumors in mice receiving control IgG (bottom left, n=6) or chimeric L3D10 (bottom right, n=5).

[0055] Figure 13 The therapeutic effect of chimeric L3D10 and 10D1 in the MC38 tumor model. Human CTLA4 knock-in mice weighing approximately 20 grams were used in the study. 1×10 6MC38 tumor cells were subcutaneously injected into Ctla4 h / h In mice, once the tumor reached a diameter of 0.5 cm, the tumor-bearing mice were randomly divided into three groups of 5 or 6 mice each. Subsequently, as indicated by the arrows, the mice were treated (ip) with 100 μg / injection of 10D1, chimeric L3D10, or control hIgGFc on days 7, 10, 13, and 16. Results from duplicate experiments are shown (left and right figures), and the data presented are the mean and standard deviation of tumor size (n = 6 / group in the left figure, n = 5 / group in the right figure). L3D10 and 10D1 showed similar therapeutic effects in this model and both induced established complete remission of the tumor. The tumor diameter (d) was calculated using the following formula: D = √(ab), V = ab² / 2, where a is the major axis and b is the minor axis. Statistical analysis was performed using two-way repeated measures ANOVA (treatment × time). In the left figure: 10D1 vs. hIgGFc: P = 5.71e-07; L3D10 vs. hIgGFc: P = 5.53e-07; 10D1 vs. L3D10: P = 0.869.

[0056] Figure 14 Anti-CTLA-4 mAb against CTLA4 h / m Effective rejection of MC38 in mice. Figure 13 That's how it is in the middle, but using hybrid CTLA4 h / m Mice. The data shown are the mean tumor diameter and SEM (6 mice / group); 10D1 vs. hIgGFc: P = 0.0011; L3D10 vs. hIgGFc: P = 5.55e-05; 10D1 vs. L3D10: P = 0.0346.

[0057] Figure 15 Therapeutic effects of chimeric L3D10 and 10D1 in a B16-F1 melanoma tumor model. Human CTLA4 knock-in mice weighing approximately 20 g were used in the study. Arrows indicate treatment time (50 μg / mouse / treatment). Data shown are the mean and standard deviation of tumor size (n = 4 / group). L3D10 showed a similar therapeutic effect in this model, and both were able to delay tumor growth in this aggressive and poorly immunogenic tumor model.

[0058] Figure 16An analysis was conducted to measure CTLA4 blockade in vivo. B7.1 or B7.2 on dendritic cells bind to and are downregulated by CTLA4 on the surface of T cells. However, blocking the binding of anti-CTLA4 antibodies prevents B7.1 / B7.2 from binding to CTLA4 and thus prevents the downregulation of both B7.1 and B7.2, resulting in a net increase in B7.1 / B7.2 expression. However, in the case of chimeric T cells expressing both human and mouse CTLA4, antibodies binding to human CTLA4 do not prevent B7.1 / B7.2 from binding to mouse CTLA4, and restore the inhibition of B7.1 / B7.2.

[0059] Figure 17 AF.10D1 does not block B7-CTLA4 interaction in vivo. (Using...) Figure 11 The analysis described herein uses cells from mice treated with anti-CTLA4 antibody to analyze the expression of B7.1 and B7.2. Figure 17 Figure A shows the experimental design. Simply put, age- and sex-matched mice received 500 μg of antibody or its control intraperitoneally. Twenty-four hours after injection, the mice were sacrificed, and their spleen cells were stained with anti-CD11c, CD11b, anti-B7-1, and anti-B7-2 mAb. Figure 17 B demonstrates CD11c expression analysis targeting B7. hi Representative data of DC phenotypes. Figure 17 Figure C shows a representative histogram depicting the levels of B7-1 on DCs from mice that received control IgG1-Fc, L3D10, or 10D1. The data in the upper figure show the antibody effect in homozygous knock-in mice, while the data in the lower figure show the antibody effect in heterozygous mice. Figure 17 D like Figure 17 It is displayed as in C, but the expression shown is from B7-2. Figure 17 The data shown in C and D represent data from 3 mice in each group, and have been repeated once, with 3 mice in each group. Figure 17 Figure E shows that in human CTLA4 homozygous mice, L3D10 but not 10D1 induces the expression of B7-1 (left panel) and B7-2 (right panel). The data shown are summarized from two experiments involving a total of 6 mice in each group. In each experiment, the mean data in the control mice were artificially defined as 100%, and the mean data in the experimental groups were normalized relative to the control. Figure 17 F such as Figure 17 As in E, but using heterozygous mice. Neither L3D10 nor 10D1 blocks B7-CTLA4 interaction in mice codominantly expressing the human Ctla4 gene.

[0060] Figure 18L3D10 binds to human, but not mouse, CTLA4. The data shown are from gated Cd3... + Cd4 + Using Ctla4 in cells h / h (above) or Ctla4 m / m (Below) Dot plot of intracellular CTLA4 staining in mouse spleen cells. Anti-mouse CTLA4 mAb 4F10 was used as a control.

[0061] Figure 19 Chip-integrated L3D10 and 10D1 in CTLA4 h / m Therapeutic effect in mice. The figure above depicts the experimental design. Ctla4... h / h Mice were challenged with the MC38 colon cancer cell line, and when the tumors reached approximately 5 mm in diameter, the mice were treated four times with control human IgG-Fc, L3D10, or 10D1, and tumor size was observed over a 6-week period. The figure below shows the growth kinetics of MC38 tumors in mice (n=6 / group) receiving control IgG, chimeric L3D10, or 10D1. Although... Figure 16 The in vivo CTLA4 blocking activities shown in the samples differed significantly, but both L3D10 and 10D1 exhibited activity against chimeric CTLA4. m / h Strong antitumor activity of the MC38 model in mice.

[0062] Figure 20 AB.10D1 and L3D10 have similar therapeutic effects on the growth of B16 melanoma. (The last part, "1×10," appears to be a separate, unrelated sentence fragment and is left untranslated.) 5 One B16 tumor cell was injected (sc) into Ctla4 h / h In mice (n=4-5), and as indicated by the arrows on days 11, 14, and 17 ( Figure 20 A) or on days 2, 5, and 8 ( Figure 20 B) Use 100μg ( Figure 20 A) or 250μg ( Figure 20 B) Treatment with 10D1, L3D10, or control IgGFc (ip). Figure 20 In A, 10D1 versus hIgGFc: P = 0.0265; L3D10 versus hIgGFc: P = 0.0487; 10D1 versus L3D10: P = 0.302. Figure 20 In group B, 10D1 versus hIgGFc: P = 0.00616; L3D10 versus hIgGFc: P = 0.0269; 10D1 versus L3D10: P = 0.370. Data represent mean ± SEM of 4–5 mice per group. Statistical analysis was performed using two-way repeated measures ANOVA.

[0063] Figure 21AB. Killed before complete rejection to assess Treg-depleted Ctla4 within the tumor microenvironment. h / h (Figure 21A) and Ctla4 m / h ( Figure 21 B) Immunotherapy effects between L3D10 and 10D1 in mice. The data shown are the mean tumor diameter and SEM from two independent experiments, with 5 mice in each group.

[0064] Figure 22 AF. Blocking B7-CTLA4 interaction does not promote the cancer immunotherapy activity of anti-CTLA4 mAb. Figure 22 A demonstrates that two anti-CTLA4 mAbs, despite significantly different blocking activities, have comparable immunotherapeutic effects. (5 × 10⁻⁶) 5 MC38 tumor cells were injected (sc) into Ctla4 h / h Mice (n=6) were treated with 100 μg 10D1, L3D10, or control hIgG-Fc on days 7, 10, 13, and 16 as indicated by arrows (ip). Data are presented as mean ± SEM for six mice per group. Statistical analysis was performed by two-way repeated measures ANOVA (treatment × time). 10D1 vs. hIgG-Fc: P = 5.71 e-07 L3D10 versus hIgG-Fc: P = 5.53e -07 ;10D1 vs. L3D10: P = 0.869. The data represent three independent experiments. Figure 22 B. In mice where neither antibody blocked B7-CTLA4 interaction, both induced stable tumor rejection. Figure 22 Similar to A, but using heterozygous mice expressing both mouse and human CTLA4. 10D1 vs. hIgG-Fc: P = 0.0011; L3D10 vs. hIgG-Fc: P = 5.55e -05 ;10D1 vs. L3D10: P = 0.0346. The data represent three independent experiments. Figure 22 CF. Blocking B7-CTLA4 interaction does not contribute to selective Treg depletion in the tumor microenvironment. Figure 22 C and D. No information regarding their ability to block B7-CTLA4 interaction; L3D10 and 10D1 did not cause Treg loss in the spleen. The data shown are for Foxp3+ cells in Ctla4... h / h ( Figure 22 C) and Ctla4 m / h ( Figure 22 D) % of CD4 T cells in the spleen of mice. n=6, e and f, L3D10 and 10D1 both contribute to CD4 T cells. h / h ( Figure 22E) and Ctla4 m / h ( Figure 22 F) Treg loss in tumor-infiltrating CD4 T cells in mice. The data shown in cf are the percentages of Treg at 17 (Experiment 1) or 19 (Experiment 2) days after tumor cell attack and at 10 or 12 days after the initial four anti-CTLA4 mAb treatments, as indicated by the arrows.

[0065] Figure 23 AF. To evaluate the blocking activity of commonly used anti-mouse CTLA4 mAb 9H10 and 9D9. Figure 23 A and B show that if B7-1 ( Figure 23 A) and B7-2 Figure 23 B) When plated, 9H10 does not block B7-CTLA4 interaction. Biotinylated mouse CTLA4-Fc fusion protein was incubated with a B7-coated plate in the presence of predetermined concentrations of control IgG or anti-mouse CTLA4 mAb 9D9 and 9H10. CTLA4 binding was detected using HRP-bound streptavidin. Data shown are the average of duplicates and represent two independent experiments. Figure 23 C and D show that 9D9 and 9H10 exhibit properties similar to soluble ( Figure 23 C) and plate combination CTLA4-Fc ( Figure 23 D) Differences in combination. The data shown are the average of duplicates and represent at least two independent experiments. Figure 23 E and F demonstrate the effect of anti-mouse CTLA4 mAb 9D9 and 9H10 on mice derived from WT 24 hours after intraperitoneal treatment with 500 μg of antibody. m / m CD11c of spleen cells hi B7-1 on DC ( Figure 23 E) and B7-2 Figure 23 The effect at the F level. Data were summarized in two independent experiments by 6 independent mice in each group, with each group involving 3 mice.

[0066] Figure 24 AD. Unique in vivo and in vivo blocking activity against mouse CTLA4 mAb 4F10. Figure 24 A and B demonstrate the effect of 4F10 on CTLA4-Fc and the board coating of B7-1 ( Figure 24 A) or B7-2 Figure 24 The effect of interaction (B) was investigated. Biotinylated mouse CTLA4-Fc fusion protein was incubated on a B7-coated plate in the presence of predetermined concentrations of control IgG or anti-mouse CTLA4 mAb 4F10. CTLA4 binding was detected using HRP-based streptavidin binding assays. Data presented are duplicated averages and represent two independent experiments. Figure 24 C and D show the effect of 4F10 on B7-1 and B7-2 expression. B7-1 (from 6 mice in each group) Figure 24 C) and B7-2 Figure 24 D) Summary data on levels. The B7 level in the control IgG treatment group was artificially defined as 100%.

[0067] Figure 25 Adverse effects of chimeric L3D10 and 10D1 in combination with anti-PD-1. The figure above depicts the experimental design. Ten-day-old female human CTLA4 knock-in mice weighing more than 4 grams were used in the study. They received the specified protein or a combination thereof. Arrows indicate the time of treatment (100 μg / mouse / treatment). The data shown are the mean and standard deviation of body weight gain % (%). Chimeric L3D10 and 10D1 showed comparable cancer therapeutic effects in adult mice (…). Figure 13 However, unique adverse effects can be observed when 10D1 is combined with anti-PD-1mAb.

[0068] Figure 26 Adverse effects of combining L3D10 and 10D1 with anti-PD-1. The figure illustrates the effects of... Figure 25 The final body weight of mice (n=5 / group) receiving control IgG, 10D1+ anti-PD-1, or chimeric L3D10+ anti-PD-1 as outlined in the experiment is shown on day 42. Significant body weight loss was observed in the anti-PD1+10D1 combination, which was not observed in the anti-PD-1+chimeric L3D10 combination.

[0069] Figure 27 The pathological effects of chimeric L3D10 and 10D1 in combination with anti-PD-1. To further examine the relative toxicity of L3D10 compared to 10D1 when administered in combination with anti-PD-1, we reviewed the above. Figure 26 Gross anatomy of mice described in the text. The uterus / ovaries / bladder and thymus of mice treated with 10D1+PD-1 were significantly smaller, while organs in mice treated with L3D10+ anti-PD-1 were comparable to those in the hIgG control. In contrast, the heart dissected from mice treated with 10D1 showed a larger size and a significantly whiter appearance.

[0070] Figure 28 AD. Treatment with a combination of 10D1 and anti-PD-1 resulted in abnormal erythrocyte production. Given... Figure 27 Observing the differences in the heart observed in mice, we examined erythrocyte production in mice and observed significant differences between mice treated with 10D1+ anti-PD-1 and groups treated with L3D10+ anti-PD-1 or a control antibody (hIgG) (which were very similar). The bone marrow of mice treated with 10D1+ anti-PD-1 was significantly whiter. Figure 28A) and the separated blood is almost completely white in color. Figure 28 B). Based on this, when we analyzed erythrocyte differentiation using the distribution of CD119 and CD71 markers, we observed a statistically significant reduction in the number of cells undergoing stage IV in mice treated with 10D1+ anti-PD-1. A representative FACS distribution map is shown below. Figure 28 In C, the summary data is presented in Figure 28 D.

[0071] Figure 29 Flow cytometry analysis of anti-erythrocyte antibodies. Blood samples from NOD.SCID.Il2rg- / - (NSG) mice were stained with plasma samples from mice that received antibody treatment during the perinatal period. Serum from NSG mice and those without serum were used as negative controls. All sera were used at a 1:50 dilution. These data showed that no mice produced anti-erythrocyte antibodies.

[0072] Figure 30 Pathology of the heart in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicology of L3D10 in combination with 10D1 and anti-PD-1, we... Figure 26 Histological analysis was performed on the hearts of the mice described in the paper. Mice treated with 10D1+ anti-PD-1 showed high levels of T cell infiltration, which was not observed in mice treated with L3D10+ anti-PD-1 or mice treated with human IgG as a control.

[0073] Figure 31 Pathology of the lungs in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicology of L3D10 in combination with 10D1 and anti-PD-1, we... Figure 26 Histological analysis was performed on the lungs of the mice described in the paper. Mice treated with 10D1+ anti-PD-1 showed high levels of T cell infiltration, which was not observed in mice treated with L3D10+ anti-PD-1 or mice treated with human IgG as a control.

[0074] Figure 32 Pathology of salivary glands in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicology of L3D10 in combination with 10D1 and anti-PD-1, we... Figure 26 Histological analysis was performed on the saliva of the mice described in the paper. Mice treated with 10D1+ antiPD-1 showed much higher levels of T cell infiltration compared with mice treated with L3D10+ antiPD-1 or mice treated with human IgG as a control.

[0075] Figure 33AF. Pathology of the kidneys and livers of mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicology of L3D10 in combination with 10D1 and anti-PD-1, we... Figure 26 Histological analysis was performed on the kidneys and livers of the mice described in the paper. Figure 33 AC is a slice from the kidney and Figure 33 DE is a slice taken from the liver. Mice treated with 10D1+ antiPD-1 showed high levels of T cell infiltration compared to mice treated with L3D10+ antiPD-1 or mice treated with human IgG as a control.

[0076] Figure 34 Toxicity fractions in mice treated with a combination of chimeric L3D10 and 10D1 and anti-PD-1. This tissue data summary is shown below. Figures 30-33 Furthermore, it demonstrates the high toxicity score of mice treated with 10D1+ anti-PD-1 relative to L3D10+ anti-PD-1, which was only slightly higher than the hIgG control group.

[0077] Figure 35 .10D1+ anti-PD-1 in Ctla4 h / m It showed no significant toxicity in mice, as evidenced by normal weight gain in mice treated with the antibody during the perinatal period. Mice were treated intraperitoneally with the predetermined antibody or combination (100 μg / mouse / injection / antibody) on days 10, 13, 16, 19, and 22. Mice were weighed at least every 3 days.

[0078] Figure 36 L3D10 and 10D1 showed similar binding patterns to plate-fixed CTLA4. ELISA plates were coated with 1 μg / ml CTLA4-His protein (Sino Biologics). Predetermined concentrations of biotinylated binding protein were added, and binding was measured using HRP-bound streptavidin. 10D1-1 and -2 are two independent batches of the same antibody. hIgG-Fc is a human Ig negative control.

[0079] Figure 37 .L3D10 exhibited reduced binding to soluble CTLA4. An predetermined concentration of anti-human CTLA4 mAb was plated and incubated overnight. After washing and blocking with bovine serum albumin, biotinylated CTLA4-Fc was added at 0.25 μg / ml. After incubation and washing, the amount of captured CTLA4-Fc was measured using HRP-labeled streptavidin.

[0080] Figure 38Alignment of the variable region of the humanized antibody with the parental L3D10 antibody sequence. The heavy chain variable region (top) (SEQ ID NO: 62-64) and light chain variable region (bottom) (SEQ ID NO: 70-72) of the humanized antibody sequence are aligned with the parental L3D10 antibody (heavy chain: SEQ ID NO: 57; light chain: SEQ ID NO: 65) and the corresponding human antibody framework (heavy chain: SEQ ID NO: 58-61; light chain: SEQ ID NO: 66-69). Reversion mutations in the mouse parental sequence are highlighted in yellow. Novel amino acids (i.e., amino acid residues not present in the parental antibody sequence or the corresponding human antibody framework) are highlighted in green. Mutations introduced into the CDR2 sequence are shown in purple. CDR sequences based on www.bioinf.org.uk / abs / are shown in red.

[0081] Figure 39 AB. Antitumor activity of humanized L3D10 antibody compared to 10D1. Using the MC38 mouse tumor model of human CTLA4 knock-in mice, we examined the antitumor activity of humanized L3D10 antibody compared to chimeric L3D10 antibody and 10D1. The top figure shows the treatment timeline for in vivo experiments; mice were administered a total of 4 doses of antibody every 3 days starting from day 7 post-inoculation. All humanized antibodies (n = 6 / group) completely eradicated tumors and were comparable to 10D1 (bottom figure).

[0082] Figure 40 Humanized L3D10 antibody in CTLA4 h / m Antitumor activity in mice. The figure above shows the treatment timeline in in vivo experiments; Ctla4 h / m Mice received either control hIg or one of three different anti-human CTLA4 mAbs at a dose of 30 (-30, solid line) or 10 (-10, dotted line) mg / injection on a specified date following MC38 tumor injection. Tumor size was measured every three days.

[0083] Figure 41 Therapeutic effect of anti-CTLA-4 mAb in a minimal disease B16-F1 tumor model. Using a B16-F1 mouse tumor model in human CTLA4 knock-in mice, we examined the antitumor activity of the humanized L3D10 antibody. 1×10 5 One B16 tumor cell was injected (sc) into Ctla4 h / hIn mice (n=5–6). Mice were treated with control Ig, 10D1, chimeric L3D10, or PP4637 and PP4638 (250 μg / mouse, ip) on days 2, 5, and 8. Tumor incidence and size were measured every other day. 10D1 vs. hIgGFc: P=0.00616; L3D10 vs. hIgGFc: P=0.0269; 10D1 vs. L3D10: P=0.370; PP4637 vs. hIgGFc: P=0.0005; PP4637 vs. 10D1: P=0.805; PP4638 vs. hIgGFc: P=0.0016; PP4638 vs. 10D1: P=0.856. Data represent mean ± SEM for 5–6 mice per group. Tumor size in mice that never developed tumors was considered 0.

[0084] Figure 42 The combination toxicity of 10D1, PP4631, and PP4637 with anti-PD-1 mAb was compared in females. As specified in the illustration, female CTLA4... h / h Mice were treated with either antibody (100 μg / mouse / injection, every three days) or control Fc injection four times on days 10 or 11 after birth. Mice were weighed every three days. The data shown are the average percentage of body weight gain (%) over a 30-day period and SEM. All mice were sacrificed on day 43 for histological analysis. The number of mice used in each group is shown in marked parentheses.

[0085] Figure 43 Combination therapy with .10D1 and anti-PD-1 resulted in anemia, while combination therapy with PP4631 + anti-PD-1 or PP4637 + anti-PD-1 did not. The data shown are hematocrit values ​​of 43-day-old mice that had received four antibody treatments at a dose of 100 μg / mouse / antibody on days 11, 14, 17, and 20.

[0086] Figure 44A The combination therapy of B.10D1 + anti-PD-1 resulted in systemic T cell activation, while the combination therapy of PP4631 + anti-PD-1 or PP4637 + anti-PD-1 did not result in systemic T cell activation. The data shown are from peripheral blood (…). Figure 44A ) or spleen ( Figure 44B ) contains the original (CD44) lo CD62L hi ), central memory (CD44) hi CD62L hi ) and effect memory (CD44) hiThe percentage of CD4 (top) and CD8 T cell phenotypes (bottom) of CD62Llo) T cells. Cells were collected from 43-day-old mice that had received antibody treatment four times at a dose of 100 μg / mouse / antibody on days 11, 14, 17, and 20.

[0087] Figure 45 Humanization of L3D10 will not affect its binding with fixed CTLA4. For example... Figure 36 The assay described above measures the ability of the humanized L3D10 antibody to bind to immobilized CTLA4. The X-axis indicates the concentration of anti-CTLA-4 mAb added to the solution. Humanization does not affect binding to immobilized CTLA4, and all three humanized antibodies exhibit similar binding to the parental chimeric L3D10 antibody and 10D1. A similar pattern was observed when CTLA4-Ig was used instead of CTLA-4-his.

[0088] Figure 46 Humanization further reduces the binding of L3D10 to soluble CTLA4. For example... Figure 37 The assay described herein measures the ability of humanized L3D10 antibody to bind soluble CTLA4. The X-axis indicates the concentration of anti-CTLA-4 mAb coated onto the ELISA plate. Humanized chimeric antibody showed a further reduction in binding to soluble CTLA4 compared to the parental L3D10 antibody. A similar pattern was observed when CTLA4-Ig was used instead of CTLA-4-his.

[0089] Figure 47 AB.PP4631, PP4638 and PP4637 do not block B7-CTLA-4 interaction in vitro. Figure 47 A demonstrates the blocking effect of anti-human CTLA-4 mAbs 10D1, PP4631, PP4637, and L3D10 on the B7-1-CTLA-4 interaction. B7-1 Fc was immobilized at a concentration of 0.5 μg / ml. Biotinylated CTLA4-Fc was added at 0.25 μg / ml along with the predetermined dose of antibody. The data shown are the average of two densities at 405 nM. Figure 47 B demonstrates the blocking of B7-2-CTLA-4 interaction by anti-human CTLA-4 mAbs 10D1 and L3D10. Figure 47 As in A, but fixed as B7-2-Fc.

[0090] Figure 48As demonstrated by their lack of effect on the expression of B7-1 and B7-2 on dendritic cells, PP4631 and PP4637 do not block B7-CTLA-4 interaction in vivo. Summary data on B7-1(a) and B7-2(b) levels from three mice in each group are presented. B7 levels in the control IgG treatment group were artificially defined as 100%.

[0091] Figure 49 Based on tumor rejection at the lowest therapeutic dose, the combination with anti-PD-1 mAb demonstrates the best safety profile (see [link]). Figure 42 PP4637 of Ctla4 is the most potent causative agent of tumor rejection. h / m Mice received either control IgFc or one of three different anti-human CTLA4 mabs at a dose of 30 (-30, solid line) or 10 (-10, dotted line) μg / injection on specified dates. Tumor size was measured every three days. At 10 μg / injection, PP4637 (HL32) induced tumor rejection most efficiently.

[0092] Figure 50 Humanized antibody purity assessment. Transiently expressed humanized L3D10 antibody was purified by protein A chromatography, and samples from all three antibodies were evaluated by reducing and non-reducing SDS-PAGE. Under both reducing and non-reducing conditions, purified proteins produced gel bands, with size indicating antibody molecule size. The "efferentiation" lanes showed flow through the protein A column, indicating that most antibody protein adhered to the protein A column.

[0093] Figure 51 Size exclusion chromatography (SE-HPLC) of transiently expressed proteins. Protein samples of each humanized antibody were analyzed by SE-HPLC using a single-step protein A chromatography method. Top image: Antibody PP4631. Middle image: Antibody PP4637. Bottom image: Antibody PP4638.

[0094] Figure 52 CE-SDS analysis of transiently expressed proteins. Protein samples from each humanized antibody were analyzed by CE-SDS using a single-step protein A chromatography method. The left panel shows results under non-reducing conditions, and the right panel shows results under reducing conditions. Top panel: Antibody PP4631. Middle panel: Antibody PP4637. Bottom panel: Antibody PP4638.

[0095] Figure 53A -C. Charge isotype distribution and deamidation of humanized L3D10 antibody as determined by capillary isoelectric focusing (cIEF). The level of protein deamidation under high pH stress was determined by comparing humanized L3D10 antibody before and after treatment with high pH stress at two different time periods (5 hours and 12.5 hours), and analyzed by cIEF. Figure 53A -C shows the distribution maps of antibodies PP4631, PP4637, and PP4638, respectively.

[0096] Figure 54 Differential scanning calorimetry (DSC) analysis of AC humanized L3D10 antibody. Differential scanning calorimetry (DSC) analysis was performed on different antibodies to determine their thermal stability and melting temperature. Figure 54 AC displays the normalized DSC curves of antibodies PP4631, PP4637, and PP4638, respectively.

[0097] Figure 55 Alignment of the extracellular domains of human, macaque, and mouse CTLA-4. The amino acid sequences of the extracellular domains of human (Hm, shown in red) (SEQ ID NO:73), macaque (Mk, shown in black), and mouse (Ms, shown in green) CTLA-4 proteins were compared, with conserved amino acids (relative to the human sequence) shown underlined (-). To aid alignment, the mouse sequence shows deletions and insertions (relative to human and monkey sequences) highlighted in yellow. Known B7-1Ig binding sites are shown in bold and underlined. The sequences show that the human and monkey sequences are highly conserved, while the mouse sequence exhibits several amino acid differences. Based on this sequence alignment, 11 mutant (M1-M11) (SEQ ID NO:40-50) human CTLA-4 Fc proteins with mouse-specific amino acids were designed, and the amino acids incorporated into each mutant protein are shown in blue.

[0098] Figure 56A - The amino acid sequences of B.WT and mutant CTLA-4Fc proteins are shown. DNA constructs encoding the WT CTLA-4Fc protein (SEQ ID NO:39) and 11 mutant proteins containing murine Ctla-4 amino acids (SEQ ID NO:40-50) are presented as shown. The amino acid sequences are for the mature protein, including the IgG1 Fc portion but excluding the signal peptide. Known B7-1Ig binding sites are shown in uppercase blue letters with double underlines. Substituted murine amino acid residues in the mutants are shown in lowercase red. The IgG1 Fc portion of the protein is underlined.

[0099] Figure 57The mutation in M11 (AA103-106, YLGI>fcGm) selectively eliminates antibody binding to human CTLA-4. The data shown are the average of duplicates, depicting the binding of B7-1Fc (a), L3D10 (b), PP4631 (c), and PP4637 (d) to plate-coated hCTLA4-Fc (hollow circle), mCTLA4-Fc (solid triangle), M11 (solid circle), and IgG1-Fc (hollow triangle).

[0100] Figure 58 In the 3D structure of the B7-1-CTLA4 complex, L3D10, PP4631, and PP4637 were mapped to epitopes adjacent to the B7-1 binding site. The B7-1 binding motif is colored red, while the antibody epitopes are colored purple. B7-1 is depicted as a space-filled band above CTLA4, while CTLA-4 is depicted as an unfilled band.

[0101] Figure 59 The amino acid sequences of .WT (SEQ ID NO:39) and mutant CTLA-4Fc proteins M12-M17 (SEQ ID NO:51-56) are shown. DNA constructs encoding six murine CTLA-4Fc amino acids, including mutant CTLA-4Fc proteins M12-M17, are presented as shown. The amino acid sequences target the mature protein, including the IgG1 Fc portion but excluding the signal peptide. Known B7-1Ig binding sites are shown in uppercase blue letters with double underlines. Substituted murine amino acid residues in the mutants are shown in lowercase red. The IgG1 Fc portion of the protein is underlined.

[0102] Figure 60 AC mutation analysis revealed 10D1 ( Figure 60 A) PP4631 ( Figure 60 B) and PP4637 Figure 60 C) Unique binding requirement with CTLA-4. CTLA-4 Fc mutants were plated overnight at 4°C at a concentration of 1 μm / ml. After BSA blockade, a predetermined concentration of biotinylated anti-CTLA-4 mAb was added and incubated for 2 hours. After washing away unbound antibodies, binding antibodies were detected using HRP-labeled streptavidin.

[0103] Figure 61 AB. Anti-4-1BB and anti-CTLA-4 antibodies in minimal disease ( Figure 61 The therapeutic effects in both A) and the established tumor (Fig. 61B) model. Figure 61 A demonstrates a treatment for minimal disease. C57BL / 6 mice were treated with 5 × 10 5MC38 cells were subcutaneously injected. On days 2, 9, and 16 post-injection, control hamsters and rats were injected with IgG, anti-CTLA-4, and / or anti-4-1BB antibodies. Tumor size was measured by physical examination. The data shown represent tumor growth kinetics, with each row representing tumor growth in one mouse species. The presented size is the product of the tumor's long and short diameters. Figure 61 B showcases established treatment options for the tumor. For example... Figure 61 As in A, but the treatment began 14 days after tumor attack; all mice had a size ranging from 9-60 mm before the start of mAb treatment. 2 The established tumor was identified. The combined effect of the two antibodies on the established tumor has been replicated three times.

[0104] Figure 62 CD8 T cells, but not CD4 or NK cells, are essential for antibody-induced tumor rejection. Tumor-carrying mice were depleted of CD4, CD8, or NK cells (*) through three injections of antibodies specific to CD4, CD8, or NK1.1 on days 9, 12, and 16 after tumor cell inoculation. Treatment antibodies (anti-CTLA-4 plus anti-4-1BB) were injected on days 9, 16, and 23 (vertical arrows). Data shown are mean tumor size and SEM (n=3). For each group, the CD8 depletion group was compared to the other groups.

[0105] Figure 63 AB. Combination therapy reduces the host response to anti-CTLA-4 antibodies. Hamster anti-mouse CTLA-4 ( Figure 63 A) or rat anti-mouse 4-1BB ( Figure 63 B) Antibodies were plated on ELISA plates. Serum from each of the five mouse groups was added to the plates at different dilutions. The relative amount of bound antibodies was determined using a second-step reagent (by absorbing biotinylated goat anti-mouse antibodies that deplete reactivity to rat and hamster IgG). The data shown are the mean optical density at 490 nm and SEM. A similar reduction in host antibody responses against CTLA-4 and 4-1BB was observed when tumor-free mice were treated with the same antibodies (data not shown).

[0106] Figure 64 AB. Combination therapy of anti-4-1BB and L3D10 (anti-human CTLA4) antibodies in human CTLA-4 gene knock-in mice. Figure 64 A demonstrates the therapeutic effect. Human CTLA4 knock-in mice were treated with 5 × 10⁻⁶ mice. 5MC38 tumor cells were subcutaneously inoculated. Two days later, groups of seven mice were treated with rat and mouse IgG, anti-4-1BB and mouse IgG, L3D10 and rat IgG, or L3D10 and anti-4-1BB, as indicated by the arrows. Data shown are mean tumor volume and SEM (n=7). All treatments significantly reduced tumor growth (P<0.001), and the double antibody treatment groups showed significantly reduced tumor size compared to the control (P<0.0001) or L3D10 antibody (P=0.0007) or anti-4-1BB antibody treatment (P=0.03). All tumor-bearing mice were euthanized when the control IgG treatment group reached the early removal criterion. Figure 64 B demonstrates durable immunity in mice receiving combination therapy. Tumor-free mice in the dual-antibody treatment group developed durable immunity against MC38 tumors. On day 110 following the first tumor cell challenge, tumor-free mice treated with the dual antibodies or untreated control mice were administered 5 × 10⁻⁶ cells / mL. 5 Subcutaneous attack with individual tumor cells. Tumor growth was monitored via physical examination. Note that all mice that rejected the tumor in the first round were fully resistant to re-attack, while all untreated mice exhibited progressive tumor growth. Detailed Implementation

[0107] definition

[0108] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule that has a "variable region" antigen recognition site. The term "variable region" is intended to distinguish this type of immunoglobulin domain from domains that are widely shared by antibodies (such as the antibody Fc domain). Variable regions contain "hypervariable regions" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from the “complementarity-determining region” or “CDR” (i.e., typically residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Ref. 44) and / or 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 residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Ref. 45). The “framework region” or “FR” residues are those variable domain residues other than those in the hypervariable region as defined herein. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camel-like antibodies, single-chain antibodies, disulfide-linked Fv (sdFv), intracellular antibodies, and anti-individual genotype (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-Id antibodies against the antibodies of the present invention). Specifically, this class of antibodies includes any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0109] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more structural residues of an antibody that contain the antibody's complementarity-determining region ("CDR") and optionally constitute the antibody's "variable region" antigen recognition site, and exhibit the ability to bind antigens with immune specificity. Such fragments include Fab', F(ab').sub.2, Fv, single-chain (ScFv) and its mutants, naturally occurring variants, and fusion proteins that contain the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., toxins, antigen recognition sites of different antigens, enzymes, receptors, or receptor ligands). As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence having 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 acid residues, 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.

[0110] Human, chimeric, or humanized antibodies are particularly preferred for in vivo use in humans; however, mouse antibodies or antibodies from other species are suitable for a wide range of uses (e.g., in vitro or in situ detection and analysis, acute in vivo use, etc.).

[0111] "Chimeric antibodies" are molecules in which different portions of an antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a constant region from 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 manufactured using a variety of techniques known in the art, including, for example, CDR transplantation (EP 239,400; International Publication WO 91 / 09967; and U.S. Patents 5,225,539, 5,530,101, and 5,585,089), inlay or surface remodeling (EP 592,106; EP 519,596; 46-48) and chain tampering (U.S. Patent 5,565,332).

[0112] This invention relates particularly to "humanized antibodies." As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more core-derivative (CDR) regions derived from non-human (typically mouse or rat) immunoglobulins. 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 "recipient." 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 more. 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 because, for instance, the entire variable region of a chimeric antibody is non-human. One approach is to say that the donor antibody has been "humanized" through a process called "humanization," because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provided the CDR. To a large extent, a humanized antibody is a human immunoglobulin (recipient antibody) whose hypervariable residues have been replaced by hypervariable residues from a non-human species (donor antibody) with the desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further optimize antibody performance. Generally, a humanized antibody will contain at least one, and typically substantially all, of the two variable domains, where all or substantially all of the hypervariable regions correspond to the hypervariable regions of a non-human immunoglobulin and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. The humanized antibody may optionally also include at least a portion of the immunoglobulin constant region (Fc) that has been altered by introducing amino acid residue substitution, deletion, or addition (i.e., mutation), and the human immunoglobulin constant region that typically binds to the Fc.γ.RIIB peptide with immune specificity.

[0113] Detailed description

[0114] Ipilimumab, an antibody targeting the human CTLA4 protein, has been shown to increase survival in cancer patients as a sole immunotherapy agent or in combination with other agents (such as, but not limited to, anti-PD-1 antibodies) (13-15). However, therapeutic effects are associated with significant adverse effects (13-18). There is a great need to develop novel anti-CTLA4 antibodies to achieve better therapeutic effects and / or fewer autoimmune adverse effects. The inventors have discovered that anti-CTLA4 antibodies can unexpectedly be used to induce cancer rejection while also reducing autoimmune adverse effects associated with immunotherapy.

[0115] This document provides antibody material compositions and their antigen-binding fragments. The invention further relates to embodiments of such molecules, wherein the molecules are monoclonal antibodies, human antibodies, chimeric antibodies, or humanized antibodies.

[0116] In detail, the present invention provides a molecule comprising an antigen-binding fragment of an antibody that specifically binds to CTLA4, and particularly human CTLA4 (preferably expressed at endogenous or transfection concentrations on the surface of living cells). Specifically, the invention relates to embodiments of such molecules, wherein the antigen-binding fragment binds to CTLA4, and wherein the living cells are T cells.

[0117] This invention relates to antibodies and their antigen-binding fragments capable of specifically binding to CTLA4. In some embodiments, such molecules are also capable of blocking the binding of B7.1 and B7.2 to CTLA4.

[0118] This invention further relates to embodiments of such molecules, wherein the molecules are monoclonal antibodies, human antibodies, chimeric antibodies, or humanized antibodies. This invention includes embodiments of such antibodies exhibiting monospecificity, bispecificity, trispecificity, or multispecificity.

[0119] The present invention further relates to embodiments of such molecules or antibodies binding to CTLA4, wherein the antigen-binding fragment comprises six CDRs, wherein the CDRs include the CDR of anti-CTLA4 antibody L3D10. Specifically, the antibody comprises three light chain and three heavy chain CDRs of anti-CTLA4 antibody L3D10.

[0120] The present invention further relates to embodiments of the above-described antibodies, wherein the antibodies are labeled as detectable or contain binding toxins, drugs, receptors, enzymes, or receptor ligands.

[0121] The present invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of any of the above-described antibody compositions and a physiologically acceptable carrier or excipient. Preferably, the composition of the present invention comprises a preventative or therapeutically effective amount of the humanized antibody of the present invention and a pharmaceutically acceptable carrier.

[0122] In one specific embodiment, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or state government, or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more precisely, for use in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or mediator administered with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, dextran solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, anhydrous skim milk, glycerin, propylene glycol, glycol, water, and ethanol. If necessary, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0123] Generally, the components of the compositions of the present invention can be supplied separately or mixed together in unit dosage forms, for example, as dry lyophilized powders or anhydrous concentrates in hermetically sealed containers (such as ampoules or capsules) indicating the active dose. When the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When the composition is to be administered by injection, an ampoule of sterile water or physiological saline for injection can be provided so that the components can be mixed prior to administration.

[0124] The compositions of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, salts formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed from cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0125] This invention further relates to the use of the antibody compositions described herein and their pharmaceutical compositions for upregulating immune responses. Upregulating the immune system is particularly important in the treatment of cancer and chronic infections, and therefore this invention can be used to treat such conditions. As used herein, the term "cancer" refers to a growth or tumor resulting from the abnormal, uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphoma. The term refers to diseases involving cells with the potential to metastasize to distant sites.

[0126] Therefore, the methods and compositions of the present invention can also be applied to the treatment or prevention of various cancers or other abnormal proliferative diseases, including (but not limited to) the following: cancers, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer; including squamous cell carcinoma; hematopoietic tumors of the lymphoid spectrum, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Berkitt lymphoma. The tumors include: lymphomas; hematopoietic tumors of the bone marrow lineage, including acute and chronic myeloid leukemia and promyelocytic leukemia; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma, and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma. Cancers resulting from apoptosis disorders are also expected to be treated by the methods and compositions of this invention. These cancers may include, but are not limited to, follicular lymphomas; cancers with p53 mutations; hormone-dependent tumors of the breast, prostate, and ovary; and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. In specific embodiments, malignant tumors or proliferative abnormalities (such as metaplasia and developmental abnormalities) or hyperproliferative disorders in the ovaries, bladder, breast, colon, lungs, skin, pancreas, or uterus are treated or prevented by the methods and compositions of the present invention. In other specific embodiments, sarcomas, melanomas, or leukemias are treated or prevented by the methods and compositions of the present invention.

[0127] In another embodiment of the invention, the antibody composition and its antigen-binding fragment can be used in conjunction with other antitumor therapies, including but not limited to current standard and experimental chemotherapy, hormone therapy, biotherapy, immunotherapy, radiation therapy, or surgery. In some embodiments, the molecules of the invention can 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 and / or prevention of cancer, autoimmune diseases, infectious diseases, or poisoning. Such agents include, for example, any of the biological response modulators, cytotoxins, antimetabolites, alkylating agents, antibiotics or antimitotic agents, and immunotherapeutic agents discussed above.

[0128] In a preferred embodiment of the invention, the antibody composition and its antigen-binding fragment can be used in conjunction with other antitumor immunotherapies. In such embodiments, the molecules of the invention are administered in combination with molecules that disrupt or enhance the activity of alternative immune regulatory pathways (such as TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, PD-1, 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 the immunomodulatory effect. Specific embodiments include bispecific antibodies comprising the anti-CTLA4 antibody composition described herein and anti-PD-1 (pembrolizumab (Keytruda or Opdivo)), anti-B7-H1 (atezilumab (Tecentriq or durvalumab), 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 another embodiment, the molecules of the present invention are administered in combination with molecules activating different stages or aspects of the immune response to achieve a broader immune response. In a more preferred embodiment, the antibody composition and its antigen-binding fragment are combined with anti-PD-1 or anti-4-1BB antibodies without exacerbating autoimmune side effects.

[0129] Another embodiment of the invention includes a bispecific antibody comprising an antibody that binds to CTLA4, which is bridged to an antibody that binds to another immunostimulatory molecule. Specific embodiments include bispecific antibodies comprising the anti-CTLA4 antibody compositions described herein and anti-PD-1, anti-B7-H1, 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. The invention further relates to the use of such antibodies for the treatment of cancer.

[0130] Methods of administering the antibody compositions of the present invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and transmucosal (e.g., intranasal and oral routes). In one specific embodiment, the antibodies of the present invention are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered via any convenient route, such as by infusion or rapid injection, absorption through the epithelial or mucosal skin lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0131] Another embodiment of the invention relates to monitoring the blocking effect of anti-CTLA4 antibodies in vivo by monitoring the expression levels of B7.1 and B7.2 on immune cells (such as antigen-presenting cells (APCs)). CTLA4 is primarily expressed in Tregs, where it suppresses autoimmune diseases by downregulating the expression of B7-1 and B7-2 on APCs (such as dendritic cells). Therefore, upregulation of B7 molecules B7.1 and B7.2 can serve as a readout for blocking B7-CTLA4 interactions in vivo. In one specific embodiment, peripheral or tumor-bound immune cells are removed from the subject before and after anti-CTLA4 treatment, and the reduction in B7.1 and / or B7.2 levels on the surface of the immune cells is analyzed ex vivo. The presence of the anti-CTLA4 blocking antibody prevents B7.1 / B7.2 from binding to endogenous CTLA4, which in turn prevents the downregulation of B7.1 and B7.2, resulting in a net increase in B7.1 / B7.2 expression. In a preferred embodiment, the levels of B7.1 and B7.1 on antigen-presenting cells are measured. In a most preferred embodiment, the levels of B7.1 and B7.1 on dendritic cells are measured.

[0132] In another embodiment, changes (reductions) in B7.1 and B7.2 on immune cells following anti-CTLA4 treatment serve as biomarkers for measuring the bioactivity of anti-CTLA4 antibodies in vivo and monitoring an apparent response to anti-CTLA4 treatment by measuring the expression levels of B7.1 and / or B7.2 on immune cells and comparing expression levels before and after treatment. In a preferred embodiment, B7.1 and / or B7.2 expression levels are monitored over time during the anti-CTLA4 treatment course.

[0133] Example

[0134] Example 1. Generation of chimeric anti-CTLA4 antibody

[0135] Using human CTLA4 knock-in mice and hu-PBL-Scid mice, previous studies have demonstrated that anti-human CTLA4 antibodies in mice reduce tumor growth, with L3D10 identified as the most effective among the tested mAbs. However, none of the antibodies obtained achieved complete tumor rejection, even at relatively high doses (>10 mg / kg) and when used after tumor cell attack before the formation of palpable tumors (e.g., early on day 2) (19-21).

[0136] Because mouse antibodies are a subclass of IgG1 that does not possess strong antibody-dependent cell cytotoxicity (ADCC), and because ADCC can participate in tumor rejection, the Fc of mAbs is modified in several ways to achieve better immunotherapeutic effects. First, mouse IgG1 with weak ADCC is replaced with human IgG1 that has strong ADCC activity to generate chimeric antibodies. Second, based on known techniques in the literature (22), three mutations (S298A, E333A, and K334A) are introduced into the CH to increase ADCC activity. Third, three mutations (M252Y, S254T, and T256E) are introduced to increase the half-life of the antibody in vivo (23). The design of the new chimeric antibodies is described in Figure 1 The image on the left.

[0137] To engineer the antibody, the variable region of the L3D10 hybridoma was first identified by DNA sequencing using standard methods known in the art. The nucleotide sequence was translated into the amino acids listed in SEQ ID NO:1 and SEQ ID NO:2. Normal human IgG1 Fc sequences and mutant Fc sequences are disclosed in SEQ ID NO:3 and SEQ ID NO:4, respectively. Amino acid and codon-optimized nucleotide sequences of the heavy and light chain sequences are disclosed in SEQ ID NO:5-8.

[0138] DNA corresponding to SEQ ID NO:5 and SEQ ID NO:7 was synthesized and inserted into an expression vector, which was then transfected into HEK293 cells with the designed sequence. In short, HEK293 cells were seeded in shake flasks one day prior to transfection and grown using a serum-free, chemically defined medium. The DNA expression construct was transiently transfected into 0.5 L of HEK293 cell suspension using standard transient transfection procedures. After 20 hours, cells were sampled to obtain viability and live cell counts, and titers (Octet QKe, ForteBio) were measured. Additional readings were taken throughout the transient transfection production run. Culture was collected on day 5. Conditioned medium of L3D10 was collected and clarified from the transient transfection production run by centrifugation and filtration. The supernatant was run on a Protein A column and eluted with a low pH buffer. Filtration was performed using a 0.2 μm membrane filter, followed by aliquoting. After purification and filtration, protein concentration was calculated from OD280 and extinction coefficient. A total of 43.2 mg of Ig protein was obtained from one round of transfection.

[0139] Example 2. The binding site of the chimeric L3D10 antibody only partially overlaps with that of 10D1.

[0140] In clinical practice, the anti-CTLA4 antibody ipilimumab has been shown to improve survival in cancer patients but induces significant adverse autoimmune effects. To evaluate the comparative binding sites of chimeric L3D10 and 10D1 antibodies, the binding to CTLA4 and the ability of antibodies to compete for binding with CTLA4 were compared. Although both antibodies bound to the immobilized CTLA4 protein with comparable efficiency ( Figure 2 However, 10D1 does not completely block the binding of chimeric L3D10 to CTLA4. Figure 3 As expected, unlabeled L3D10 completely blocked the binding of labeled L3D10, indicating that the antibody binding sites of L3D10 and 10D1 only partially overlap.

[0141] Example 3. Chimeric L3D10 antibody blocks the interaction between CTLA4:B7.1 and CTLA4:B7.2 more effectively than 10D1.

[0142] It has been reported that anti-human CTLA4 mAb 10D1 can block B7-CTLA4 interaction if soluble B7-1 and B7-2 are used to interact with immobilized CTLA4 (49). Since B7-1 and B7-2 act as cell surface co-stimulatory molecules, we used immobilized B7-1 and B7-2 to assess the ability of anti-CTLA4 antibodies to block B7-CTLA4 interaction. Using a competitive ELISA assay, L3D10 and 10D1 blocked the ability of the CTLA4 fusion protein CTLA4-Ig to bind to both plate-immobilized and cell membrane-expressed B7.1 and B7.2. In these experiments, chimeric anti-human CTLA4-mAbs with affinity (2.3 nM) similar to 10D1 (4 nM) (49) were used. In the plate immobilization assay, B7.1Fc or B7.2Fc was plated at 1 μg / ml onto ELISA plates and incubated overnight at 4°C or for 2 hours at 37°C. Biotinylated CTLA4-Fc was mixed with predetermined concentrations of B7.1-Fc, 10D1, or chimeric L3D10. The amount of CTLA4-Fc bound to the plate was determined using horseradish peroxidase-conjugated streptavidin. Figure 4 As shown, while the chimeric L3D10, B7.1Fc, and CTLA4-Fc all efficiently blocked the CTLA4-Fc:B7.1 interaction, two individual batches of 10D1 failed to block the interaction. L3D10 exhibited significant blocking of plate-immobilized B7.1 binding at concentrations as low as 0.2 μg / ml, achieving 50% inhibition (IC50) at approximately 3 μg / ml. 50 Similarly, L3D10 was used at an IC50 concentration of 0.03 μg / ml. 50 Blocking the binding of CTLA4-Fc to plate fixation B7.2, while 10D1 from two different material batches at approximately 200 μg / ml IC50 Showing minimal blockage ( Figure 5 However, consistent with previous reports (49), antibody 10D1 potently inhibited the B7-1-CTLA4 interaction in reverse experiments when plate-immobilized CTLA4 was used to interact with soluble B7-1. Figure 6 ).

[0143] In cell membrane protein binding assays, L3D10 blocked CTLA4-Fc binding when B7.1 was expressed on the surface of CHO cells, but 10D1 from two different material batches did not block the binding even when used at 512 μg / ml. Figure 7 Although far less potent than L3D10, high doses of 10D1 achieved approximately 25% blockade between human CTLA4 and mouse B7-1. Figure 8 For B7.2 expressed on the surface of CHO cells, L3D10 again blocked the expression, while 10D1 only partially blocked it, with less than 50% inhibition observed even when 10D1 was used at 512 μg / ml. Figure 9 ).

[0144] A potential caveat is that biotinylation may affect the binding of 10D1 to CTLA4-Fc. To address this, we compared the binding of L3D10 and 10D1 to biotinylated CTLA4-Fc used in the blocking studies. Figure 10 As shown, 10D1 is more effective than L3D10 in binding biotinylated CTLA4-Fc. Therefore, the failure of 10D1 to block is not due to insufficient binding to biotinylated CTLA4-Fc. A similar pattern was observed when multihistidine-labeled CTLA4 was used to interact with human B7-1 transfected CHO cells. Figure 11 In summary, our data indicate that the ability of antibody 10D1 to block B7-CTLA4 interaction is highly dependent on the analysis used; if B7-1 and B7-2 are immobilized, there is minimal to undetectable blocking activity, while antibody L3D10 is a stable blocker of B7-CTLA4 interaction, regardless of whether the B7 protein is immobilized.

[0145] Example 4. Chimeric L3D10 antibodies induce tumor rejection more efficiently than unmodified L3D10.

[0146] Previous reports indicated that L3D10 in mice failed to induce complete remission of MC38 tumors, even with a significant delay observed (19,20). To determine whether chimeric L3D10 could induce complete remission in syngeneic mice, 1×10⁻⁶ L3D10 was used to induce complete remission in syngeneic mice. 6One MC38 tumor cell was transplanted into syngeneic C57BL / 6 mice. One week later, when the tumor reached approximately 5 mm in diameter, the mice were treated with control IgG or chimeric L3D10 mAb at half the dose used in previous studies in the L3D10 case in mice. Figure 12 As shown, despite the potential immunogenicity of the human Ig sequence, chimeric L3D10 was found to result in complete remission in all tested mice. Because treatment is initiated when the tumor burden is established, which is much more difficult than when the tumor is inaccessible (19), these experiments show that chimeric L3D10 is more effective than unmodified L3D10.

[0147] Example 5. Chimeric L3D10 antibodies and 10D1 have equivalent activity in inducing tumor rejection.

[0148] The availability of human CTLA4 knock-in mice (20) provides an unprecedented opportunity to test the bioactivity of chimeric anti-human CTLA-4 antibodies using clinically available anti-CTLA-4 mAb 10D1. In this humanized mouse model, the CTLA4 gene, encoding a product with 100% identity to the human CTLA-4 protein, is expressed under the control of the endogenous mouse CTLA4 locus. When the antitumor activity of chimeric L3D10 and 10D1 was directly compared in the MC38 tumor model in human CTLA4 knock-in mice, it was evident that both antibodies were comparable in inducing tumor rejection, while tumors progressively grew in the IgG control group. Figure 13 The results of antibody treatment on tumor size are shown in duplicate in the experiment.

[0149] An interesting question is whether anti-CTLA-4 mAbs need to interact with all CTLA-4 molecules (i.e., achieve target saturation) to exert their immunotherapeutic effect. (From CTLA4) h / h and CTLA4 m / m The F1 mice co-dominantly expressed both mouse and human CTLA-4 proteins. Interestingly, as... Figure 14 As shown, both chimeric L3D10 and 10D1 effectively induced tumor rejection, even though approximately 50% of the CTLA-4 protein (i.e., the murine form of the protein) failed to bind to the anti-human CTLA-4 mAb. Importantly, in this setting, i.e., when the gene dose was limited, L3D10 was more effective than 10D1 in treatment (P<0.05).

[0150] Previous studies have confirmed that anti-mouse CTLA-4 mAb fails to induce rejection in the B16-F1 melanoma cell line without combination with other treatment modalities. Therefore, the antitumor effects of chimeric L3D10 and 10D1 antibodies were also tested using this more challenging B16 tumor model in human CTLA4 knock-in mice. Figure 15 As shown, neither L3D10 nor ipilimumab caused rejection of established tumors, but both caused statistically significant delays in tumor growth, while the differences between the different antibodies were not statistically significant.

[0151] Example 6: In vivo CTLA4 blockade

[0152] CTLA4 is primarily expressed in Treg cells, where it suppresses autoimmune diseases by downregulating the expression of B7-1 and B7-2 on dendritic cells (50). Because targeted mutations of CTLA4 (50) and treatment with blocking anti-CTLA4 mAbs (51) upregulate the expression of B7-1 and B7-2 on dendritic cells, it has been shown that the physiological function of CTLA4 on Treg cells is to downregulate B7 on dendritic cells. Therefore, B7 upregulation acts as a readout to block the B7-CTLA4 interaction in vivo, and CTLA4 cells with homozygous knock-in of the human CTLA4 gene are used. h / h An analysis was developed for mouse T cells.

[0153] like Figure 16 As outlined in the literature, surface-expressed B7.1 or B7.2 binds to CTLA4 on the surface of T cells, leading to a downregulation of B7.1 and B7.2 expression. However, blocking the binding of anti-CTLA4 antibodies prevents B7.1 / B7.2 binding, which in turn prevents the downregulation of B7.1 and B7.2, resulting in a net increase in B7.1 / B7.2 expression. However, in the case of chimeric T cells expressing both human and mouse CTLA4, antibodies binding to human CTLA4 do not prevent B7.1 / B7.2 from binding to mouse CTLA4, and restore the inhibition of B7.1 / B7.2.

[0154] Humanized CTLA4 mice, expressing a CTLA4 gene with 100% identity to the human CTLA4 protein under the control of the endogenous mouse CTLA4 locus (20), have been described. Homozygous knock-in mice (CTLA4...) h / h Backcross with C57BL / 6 background to obtain at least 10 generations. By making CTLA4 h / h Mice were crossed with WT BALB / c mice to produce heterozygous mice (CTLA4). h / m ).

[0155] To test the clinically proven therapeutic efficacy of anti-CTLA4 mAb 10D1, we injected an extremely high dose of anti-CTLA4 mAb (500 μg / mouse, which is approximately 25 mg / kg or 8 times the highest dose used clinically) into CTLA4. h / h Or Ctla4 m / h In mice, spleen cells were collected 24 hours after injection to measure Cd11c. hi The levels of B7-1 and B7-2 on DC ( Figure 17 AB). For example Figure 17 As shown in CE, compared with Ctla4 which receives human IgG1-Fc h / h Compared to mice, B7.1 expression in DCs from chimeric L3D10-treated mice was statistically significantly increased in T cells expressing human CTLA4, but not statistically significantly increased in T cells expressing both human and mouse CTLA4. Figure 17 As shown in CE, similar results were observed for B7.2. The upregulation of B7-2 was comparable to that achieved in human Treg-DC cocultures using blocking anti-CTLA4 mAb (66).

[0156] To further confirm the specificity of the in vivo analysis, we tested whether L3D10 could upregulate CTLA4 co-dominant expression in mice and humans. m / h B7 in mice. Because at least 50% of CTLA4 does not bind to anti-human CTLA4 antibodies, they are not expected to be very potent in blocking B7-CTLA4 interactions. In fact, neither antibody induces B7-CTLA4 interaction. m / h Upregulation of B7-1 and B7-2 on mouse DCs ( Figure 17 C, D, F). Ctla4 m / h The complete absence of L3D10 blockade in mice indicates that the gene encoded by the mouse allele does not bind to L3D10 ( Figure 18 CTLA4 is sufficient to downregulate B7 expression. Therefore, our data confirm that at doses at least 8 times higher than the highest dose used clinically, 10D1 does not block B7-CTLA4 interaction in vivo or in vitro when B7 is immobilized on the plate or anchored to the cell membrane.

[0157] Ctla4 m / h The complete absence of L3D10 blockade in mice indicates that the gene encoded by the mouse allele does not bind to L3D10 ( Figure 18 CTLA4 levels were sufficient to downregulate B7 expression. In contrast, 10D1 did not increase B7.1 or B7.2 expression. According to the model, this suggests that L3D10 blocks CTLA4 activity in vivo, while 10D1 does not block said activity.

[0158] However, despite these significant differences in blocking activity, both L3D10 and 10D1 exhibited activity against chimeric CTLA4. m / h The strong antitumor activity of the MC38 model in mice, such as Figure 19 As shown in the figure. Although tumors progressively grew in control Ig-treated mice, complete rejection could be achieved with either anti-CTLA4 mAb. In multiple experiments, the two antibodies were comparable in inducing tumor rejection. In another tumor model, B16 melanoma, both antibodies induced similar tumor growth delay, but complete rejection could not be achieved with either antibody. Figure 20 ).

[0159] Example 7: Antitumor effect is associated with depletion of intratumoral Tregs

[0160] In vivo immune regulation arises from the balance between immune cell activation and immune checkpoints. Specifically, regulatory T cells (Tregs) are a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and eliminate autoimmune diseases. Recent studies have demonstrated that the therapeutic efficacy of anti-mouse CTLA4 mAb is influenced by Fc subclasses and host Fc receptors, which in turn selectively affect antibody-dependent cytotoxicity of Tregs within the tumor microenvironment (52,53). Because differential CTLA4 blocking activity in vivo does not appear to translate into differential antitumor activity, we attempted to establish the mechanism of action of antitumor development and examine Tregs within the tumor microenvironment. To do this, we sacrificed mice carrying MC38 tumors before rejection was completed ( Figure 21 And analyze Ctla4 who received control Ig, 10D1 or L3D10 h / h The frequency of Tregs in knock-in mice. Although neither antibody reduced the number of Tregs in the spleen. Figure 22 C), but both reduce Treg (C) in the tumor microenvironment. Figure 22 E). Interestingly, 10D1, but not L3D10, amplifies Tregs in the spleen. Treg amplification in the spleen induced by 10D1 replicates the clinical findings of ipilimumab increasing FOXP3 expression in peripheral blood leukocytes (54). Because blocking and non-blocking antibodies are comparable in terms of Treg depletion in the tumor microenvironment, blocking B7-CTLA4 interaction does not contribute to Treg depletion. This is because 10D1 does not block B7-CTLA4 interaction in vivo and also affects Ctla4... h / hThe therapeutic effect was observed in mice and melanoma patients, so blocking this interaction was not necessary for its therapeutic effect. Furthermore, because the two mAbs with significantly different blocking effects had comparable therapeutic effects and selective Treg depletion in the tumor microenvironment, blocking the CTLA4-B7 interaction did not enhance the antibody's therapeutic effect.

[0161] To confirm this observation, we tested two anti-CTLA4 mAbs on Ctla4 m / h The therapeutic effects in mice, where anti-human CTLA4 mAbs could bind to up to 50% of CTLA4 molecules and neither antibody blocked B7-CTLA4 interaction to achieve upregulation of B7 on dendritic cells. Figure 16 Furthermore, both antibodies led to rapid rejection of MC38 tumors, but L3D10 was slightly more effective than 10D1. Figure 22 B). Accordingly, both antibodies selectively depleted Tregs (22E and 22F) in the tumor microenvironment. These genetic data further confirm that tumor rejection CTLA4 blockade is not related to local Treg depletion, and thus refute the popular hypothesis that anti-CTLA4 mAbs induce cancer immunity by blocking B7-CTLA4 interactions (10).

[0162] Example 8. Evaluation of the blocking activity of commonly used anti-mouse CTLA4 mAb 9H10 and 9D9

[0163] Based on the stimulatory effects of two anti-mouse CTLA4 mAbs (30,31) 4F10 and 9H10 on the whole and Fab, the concept of CTLA4 as an intrinsic negative regulator of T cell regulation has been proposed, but no data have been presented to confirm that these antibodies block B7-CTLA4 interaction. Recently, a third anti-mouse CTLA4 mAb, 9D9, has been reported to have therapeutic effects in tumor-bearing mice and to cause local Treg depletion in the tumor microenvironment (52). We therefore set out to test the ability of all three commercially available anti-mouse CTLA4 mAbs that have shown to induce tumor rejection to block B7-CTLA4 interaction in physiologically relevant configurations. As a first test, we used increased amounts of anti-CTLA4 mAb (up to 2,000-fold molar excess relative to CTLA4-Fc) to block the binding of biotinylated CTLA4-Fc to plate-fixed B7-1 and B7-2. Figure 23 As shown in A, anti-mouse CTLA4 mAb 9H10 did not block B7-1-CTLA4 interaction even at the highest tested concentration, but moderate blocking was observed when 9D9 was used at extremely high concentrations. While mAb 9D9 effectively blocked B7-2-CTLA4 interaction, 9H10 failed to block it. Figure 23B). Interestingly, while 9D9 exhibited strong binding to soluble CTLA4-Fc, 9H10 showed poor binding (B). Figure 23 c), even though it is more potent than 9D9 in binding to CTLA4-Fc in fixed mice ( Figure 23 D). Because the lack of any blocking activity of 9H10 in this analysis could simply reflect its poor binding to soluble CTLA4-Fc, we again used WT mice (CTLA4-Fc). m / m The upregulation of B7-1 and B7-2 on dendritic cells was used to measure the in vivo blockade of B7-CTLA4 interaction. Figure 23 As shown in E and F, 9H10 did not regulate B7-1 expression on DCs, while 9D9 increased B7-1 levels by 15% (P<0.05). Interestingly, while 9D9 explicitly upregulated B7-2 on DCs, 9H10 did not. Therefore, the first and most extensively studied tumor immunotherapy anti-CTLA4 mAb 9H10 does not block B7-CTLA4 interaction. Thus, blocking B7-CTLA4 interaction does not contribute to the induction of anti-tumor immunity via anti-mouse CTLA4 mAb. Since both mAbs exhibited considerable immunotherapy effects and considerable Treg loss in the tumor microenvironment (52), local Treg loss rather than blocking B7-CTLA4 interaction provides a unified explanation for the therapeutic effect of anti-mouse CTLA4 mAb. Interestingly, while 4F10 blocked B7-CTLA4 interaction in vitro, it failed to induce upregulation of B7 on DCs in vivo (P<0.05). Figure 24 ).

[0164] In summary, we have demonstrated that clinically proven therapeutic anti-human CTLA4 mAb (10D1) and two anti-mouse CTLA4 mAbs (9H10 and 4F10) confer immunotherapeutic effects under physiologically relevant conditions without blocking B7-CTLA4 interaction. Furthermore, this type of blockade is not necessary for tumor rejection, even for mAb (L3D10) which can potently block B7-CTLA4 interaction. Since the therapeutic effects are largely similar for antibodies that differ by a 1000-fold in blocking B7-CTLA4 interaction, this type of blockade does not contribute to the cancer therapeutic effect of anti-CTLA4 mAbs. These data refute the hypothesis that anti-CTLA4 mAbs confer immunotherapeutic effects through checkpoint blockade (55). By refuting the prevailing hypothesis, our data suggest that the therapeutic effect of anti-CTLA4 mAbs cannot be optimized by improving their blocking activity. In this context, it is particularly interesting to note that tremelimumab, which is superior in blocking B7-CTLA4 interaction (56), failed to meet its clinical endpoint in a phase III clinical trial (57). Meanwhile, by demonstrating a strong correlation between tumor rejection and local Treg depletion and by refuting the involvement of blocking B7-CTLA4 interaction in tumor immunity, our study promotes the hypothesis that local Treg depletion within the tumor environment is the primary mechanism of therapeutic anti-CTLA4 mAbs, and thus proposes new approaches for developing next-generation anti-CTLA4 mAbs for cancer immunotherapy.

[0165] Ultimately, the accumulated genetic data in mice suggest that the initial concept (30,31) of CTLA4 negatively regulating T cell activation and that this regulation is achieved through SHP-2 (58,59) may need to be reconsidered (60). Therefore, although CTLA4 - / - Severe autoimmune disease in mice has been used to support the idea that CTLA4 is an intrinsic negative regulator of T cell activation (61,62), but at least three lines of genetic data have emerged that contradict this view. First, lineage-specific deletion of the Ctla4 gene in Tregs but not effector T cells is sufficient to reproduce the autoimmune phenotype observed in germline mice with Ctla4 gene deletion (50). These data suggest that Ctla4... - / - Autoimmunity in mice is not due to a lack of the intrinsic negative regulator CTLA4 in effector T cells. Secondly, in mice composed of WT and CTLA4... - / -In chimeric mice composed of both WT and T cells, the autoimmune phenotype was prevented by the coexistence of WT T cells (63). These data strongly suggest again that autoimmune diseases are not caused by a lack of intrinsic negative regulators. The effect of a lack of intrinsic negative regulators is further demonstrated by the fact that Ctla4 was not observed during viral infection in chimeric mice. - / - Preferential expansion of T cells (64). Third, T cell-specific loss of Shp2, which is proposed to mediate negative CTLA4 regulation (58,59), has been shown to reduce rather than enhance T cell activation (65). In the context of these reported genetic data, and given the proposed negative regulator of T cell activation of CTLA4, our data reported here call for a reassessment of CTLA4 checkpoint blockade in cancer immunotherapy.

[0166] Example 9. Chimeric L3D10 exhibits reduced adverse immune events when used in combination with other immunotherapy antibodies.

[0167] Recent clinical studies have demonstrated that combination therapy between anti-PD-1 and anti-CTLA4 mAbs further increases survival in patients with advanced melanoma. However, 55% of patients receiving combination therapy experience grade 3 and 4 immune-related adverse events (irAEs). Therefore, developing antibodies with lower toxicity is crucial. We have developed an in vivo model that reproduces the irAEs observed clinically with combination therapy of anti-CTLA-4 and anti-PD-1 mAbs. In this model, we treated perinatal human CTLA4 knock-in mice (CTLA4) with high doses of anti-PD-1 and anti-CTLA-4 mAbs. h / h We found that while young mice tolerated treatment with mAb alone, the combination therapy of anti-PD-1 and 10D1 led to severe irAEs with multi-organ inflammation, anemia, and other adverse events. Figure 25 The severe growth arrest shown is illustrated in the image. In contrast, when combined with anti-PD-1, chimeric L3D10 exhibits only mild irAE as seen with normal weight gain.

[0168] To further examine the relative toxicity of chimeric L3D10 compared to 10D1 when administered in combination with anti-PD-1, we examined CTLA4 levels at 42 days post-administration. h / h Pathological effects in knock-in mice. For example... Figure 26As shown, the final body weight (day 42) of mice treated with L3D10+ anti-PD-1 was similar to that of mice treated with the hIgG negative control antibody. However, by comparison, the body weight of mice treated with 10D1+ anti-PD-1 was significantly lower. Therefore, when we examined the gross anatomy of these mice, the uterus / ovaries / bladder and thymus of mice treated with 10D1+ PD-1 were significantly smaller (Fig. 27). Furthermore, the organs of mice treated with L3D10+ anti-PD-1 were comparable to those of the hIgG control. In contrast, the heart dissected from mice treated with 10D1 showed a slightly larger size and a significantly whiter appearance. Therefore, we decided to examine erythropoiesis in the mice and observed a significant difference between mice treated with 10D1+ anti-PD-1 and the groups treated with L3D10+ anti-PD-1 or the control antibody (which were extremely similar). Figure 28 As shown in A, the bone marrow of mice treated with 10D1+ anti-PD-1 was significantly whiter, and the separated blood was almost completely white. Figure 28 b). Based on this, when we use CD71 and CD119 markers to examine cells undergoing different stages of hematopoiesis more closely, a representative FACS distribution map is shown. Figure 28 In C, the summary data is presented in Figure 28 In D, these data show a statistically significant reduction in the number of cells undergoing phase IV in mice treated with 10D1+ anti-PD-1. Figure 28 D).

[0169] To explore the potential mechanism of anemia in mice treated with 10D1, we tested whether 10D1+PD-1 treatment induced anti-erythrocyte antibodies. Figure 29 As shown, no anti-erythrocyte antibodies were detected. Therefore, the presence of erythrocyte-specific autoantibodies did not cause anemia in mice treated with anti-PD-1+10D1.

[0170] To further determine the toxicology of L3D10 in combination with 10D1 and anti-PD-1, we conducted cardiac studies after immobilization in 10% formalin for at least 24 hours. Figure 30 ),lung( Figure 31 ), salivary glands ( Figure 32 ) as well as the kidneys and liver ( Figure 33 Histological analysis was performed. In each tissue studied, mice treated with 10D1+ anti-PD-1 showed high levels of T-cell infiltration. Toxicity scores based on inflammatory severity were summarized in... Figure 34 The study showed that mice treated with 10D1+ anti-PD-1 had a high toxicity score relative to L3D10+ anti-PD-1, which was only slightly higher than the hIgG control group.

[0171] Example 10: L3D10 has reduced binding to soluble CTLA4.

[0172] L3D10 and 10D1 are fixed to the CTLA4 plate, demonstrating a similar combination mode. Figure 36 As a possible explanation for the reduced toxicity of L3D10 relative to 10D1, specifically the increased T-cell infiltration / activity associated with 10D1, we decided to examine its binding to soluble CTLA4. We chose to examine this because the association between CTLA4 polymorphisms and various autoimmune diseases involves the generation of defects in soluble CTLA4 (Nature 2003, 423:506-511), and genetic silencing of the sCTLA4 isotype increases the onset of type 1 diabetes in mice (Diabetes 2011, 60:1955-1963). Furthermore, soluble CTLA4 (abatacept and belapatacept) are widely used immunosuppressive drugs. Following this idea, when we examined the relative binding to soluble CTLA4, we observed a significant reduction in L3D10 binding (…). Figure 37 ).

[0173] We have confirmed that anti-CTLA-4 mAb in heterozygous CTLA4 h / m In mice, stable tumor injection was induced, with only 50% of CTLA-4 molecules binding to anti-human CTLA-4 mAbs. To determine whether 50% CTLA-4 involvement was sufficient to induce irAE, we treated CTLA4 with anti-PD-1+10D1. h / m Mice. For example... Figure 35 As shown, anti-PD-1+10D1 did not induce Ctla4. h / m Weight loss in mice. Therefore, irAE and cancer immunity can be genetically uncoupled.

[0174] In vivo activity confirmed that the L3D10 antibody retained its antitumor activity but exhibited reduced autoimmune adverse events compared to those observed with other immunotherapeutic antibodies (such as 10D1), suggesting the potential to enhance antitumor activity without exacerbating autoimmune adverse events. Therefore, autoimmune side effects are not a necessary cost of cancer immunity and it is possible to uncouple these two activities. Characterization of L3D10 confirmed that its ability to block the interaction of CTLA4 with B7.1 and B7.2 is more effective than that of 10D1, and this involves differences in the CTLA4 binding sites between the antibodies. Furthermore, L3D10 fused with a mutated modified human IgG1 Fc domain possessing strong ADCC activity that confers enhanced antibody therapeutic effects. Further characterization confirmed that L3D10 and 10D1 bind to immobilized CTLA4 with similar binding profiles. However, L3D10 exhibited a significantly lower binding affinity to soluble CTLA4 than 10D1. Taken together, our data confirm that the antibody L3D10 has great potential for clinical use in treating cancer patients with fewer serious adverse events.

[0175] Example 11. Humanization of L3D10

[0176] The humanization process begins with the generation of homology-modeled antibody 3D structures and the creation of a distribution of parental antibodies based on structural modeling. The receptor framework to be utilized is identified based on overall sequence consistency across the framework, matching interface locations, typical CDR locations of similar classes, and the presence of N-glycosylation sites that must be removed. A light chain (LC) and a heavy chain (HC) framework were selected for the humanization design.

[0177] Humanized antibodies are designed by creating multiple hybrid sequences that fuse selected portions of the parental antibody sequence with human framework sequences (including the transplantation of CDR sequences into the receptor framework). The predicted CDR sequences of the parental antibody L3D10 are provided as SEQ ID NO:21-26 in Table 1A below:

[0178] Table 1A: Predicted CDR sequence of parental antibody L3D10

[0179] antibody chain CDR SEQ ID NO Variable light 1 21 2 22 3 23 Variable weight 1 24 2 25 3 26

[0180] Using 3D models, both visual and computer-aided modeling, these humanized sequences were systematically analyzed to isolate the sequences most likely to retain antigen binding. The goal was to maximize the amount of human sequences in the final humanized antibody while preserving the specificity of the initial antibody.

[0181] Three humanized light chains (LC1, LC2, and LC3) and three humanized heavy chains (HC1, HC2, and HC3) were designed based on the selected receptor framework. Each of the three HC or three LC sequences originated from the same germline, such as... Figure 38The reverse mutations of the mouse parental sequences shown are different. The humanized variable region amino acid sequences and their optimized coding nucleotide sequences are listed in SEQ ID NO: 9-20. The CDR2 sequences of the humanized heavy and light chains contain amino acid changes relative to the parental L3D10 antibody sequences and are listed in SEQ ID NO 33-38 as indicated in Table 1B below.

[0182] Table 1B: CDR2 sequence of the variable region of humanized antibody.

[0183] antibody sequence CDR2 sequence SEQ ID NO HC1 YIWYDGNTNFHPSLKSR 33 HC2 YIWYDGNTNFHSSLKSR 34 HC3 YIWYDGNTNFHSPLKSR 35 LC1 AATNLQS 36 LC2 AATNLQD 37 LC3 AATSLQS 38

[0184] The light and heavy humanized chains can now be combined to form variant fully humanized antibodies. The expression levels and antigen-binding affinity of all possible combinations of humanized light and heavy chains are tested to identify antibodies that behave similarly to the parental antibody.

[0185] A novel tool for calculating the humanization score of monoclonal antibodies was used (24). This score represents the degree to which the antibody's variable region sequence resembles a human, which is an important factor in antibody humanization. The humanization scores of parental and humanized antibodies are shown in Tables 2 and 3 below. Based on our method, a score of 79 or higher indicates a human-like appearance for the heavy chain and 86 or higher for the light chain.

[0186] Table 2: Humanized Light Chain Information and Humanization Score.

[0187]

[0188] Table 3: Humanization Relink Information and Humanization Score.

[0189]

[0190] The full-length antibody gene was constructed as follows: First, variable region sequences were synthesized. Sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into expression vectors already containing human Fc domains; for the heavy chain, the hIgG1 (M252Y, S254T, T256E, S298A, E333A, K334A) backbone was used. Additionally, for comparison, the variable regions of the chimeric parental heavy and light chains were constructed into a full-length chimeric chain using the same backbone Fc sequence.

[0191] All nine humanized antibodies underwent small-scale production in 0.01 liters. Chimeric parental antibodies were also scaled up for direct comparison. Plasmids specifying the heavy and light chains were transfected into HEK293 cell suspensions in the absence of serum using chemically defined media to obtain antibodies. Whole antibodies in conditioned media were purified using MabSelect SuRe protein A medium (GE Healthcare). The 10 tested antibodies are shown in Table 4 below.

[0192] Table 4: Ten antibodies transiently produced in HEK293 cells

[0193] Antibody name Heavy chain Light chain PP number Yield (mg / L) Humanized HC1+LC1 H3106 L3106 4630 54 Humanized HC1+LC2 H3106 L3107 4631 50 Humanized HC1+LC3 H3106 L3108 4632 45 Humanized HC2+LC1 H3107 L3106 4633 37 Humanized HC2+LC2 H3107 L3107 4634 44 Humanized HC2+LC3 H3107 L3108 4635 40 Humanized HC3+LC1 H3108 L3106 4636 46 Humanized HC3+LC2 H3108 L3107 4637 55 Humanized HC3+LC3 H3108 L3108 4638 53 Chimeric Parents H2872 L2872 4629 28

[0194] The affinity of nine humanized antibody combinations and chimeric parental antibodies for the antigen (huCTLA4) was evaluated using Octet. Multi-concentration kinetic experiments were performed on the Octet Red96 system (ForteBio). The anti-hIgG Fc biosensor (ForteBio, serial number 18-5064) was hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) and pre-adjusted at pH 1.7 glycine. The antigen was diluted using a 7-point, 2-fold serial dilution, starting at 600 nM with the sample diluent. All antibodies were diluted to 10 μg / mL with the sample diluent and subsequently immobilized onto the anti-hIgG Fc biosensor for 120 seconds. After establishing a baseline in the sample diluent for 60 seconds, the biosensor was moved to wells containing a series of antigen concentrations to measure association. For each target protein in the sample diluent, association was observed for 120 seconds and dissociation for 180 seconds. Binding affinity was characterized by fitting kinetic sensor plots to a monovalent binding model (1:1 binding). Full kinetic measurements are summarized in Table 5 below.

[0195] Table 5: Kinetic measurements of humanized antibodies and parental antibodies

[0196]

[0197]

[0198] Example 12. Antitumor activity of humanized anti-CTLA4 antibody

[0199] Based on relative binding affinity and humanity score, we selected three antibodies for further evaluation:

[0200] PP4631 - High affinity and good expression

[0201] PP4637 - High affinity and good expression

[0202] PP4638 - Slightly lower affinity but highest humanization score

[0203] Materials for each of these antibodies were produced by transiently producing protein A in HEK293 cells at a scale of 0.1 L followed by purification. The binding affinity of the purified antibodies was confirmed by Octet analysis, as shown in Table 6 below.

[0204] Table 6. Kinetic measurements of humanized antibodies and parental antibodies

[0205]

[0206] We used the syngeneic MC38 mouse tumor model in human CTLA4 knock-in mice as described in Example 5 above to evaluate the antitumor activity of these three humanized antibodies compared to the 10D1 and chimeric L3D10 antibodies. Figure 39 A shows the treatment timeline for the in vivo experiment; mice were given a total of four doses of antibody every three days, starting on day 7 post-inoculation. Figure 39 As shown in B, all humanized antibodies completely eradicate tumors and are comparable to 10D1.

[0207] In another experiment, we used the heterozygous Ctla4 described in Example 5. h / m mice ( Figure 14 In the syngeneic MC38 mouse tumor model, the antitumor activity of humanized antibodies PP4631 and PP4637 was evaluated at two different doses compared to 10D1 and chimeric L3D10 antibodies. Figure 40 As shown, all mAbs were indistinguishable when used at 30 mcg / mouse / injection (1.5 mg / kg), while PP4637 was more effective at 10 mcg / mouse / injection (0.5 mg / kg), and PP4631 and 10D1 showed comparable activity.

[0208] For example Figure 41 The diagram shows the antitumor activity of the humanized antibody compared to 10D1 and chimeric L3D10 antibodies, demonstrated using a B16-F1 melanoma mouse tumor model derived from human CTLA4 knock-in mice. Mice were administered a total of three doses of the antibody every three days, starting on day 2 post-inoculation. Figure 41 As shown, L3D10 and humanized antibodies delayed tumor growth and were comparable to 10D1.

[0209] Example 13. Humanized clones of L3D10 maintain a superior safety profile compared to 10D1.

[0210] To test whether the superior safety profile of L3D10 is maintained after humanization, we compared PP4631 and PP4637 with 10D1 regarding their adverse effects when used in combination with anti-PD-1 inhibitors. Figure 42 As shown, PP4631 and PP4637 have less than 10D1 toxicity when used in combination with anti-PD-1.

[0211] and Figure 28 The defective red blood cell production described in the text is consistent with that, such as Figure 43 As shown, based on complete blood cell counts (CBCs), mice treated with 10D1 plus anti-PD-1 were anemic, while mice receiving anti-PD-1+PP4631 and anti-PD-1+PP4637 had essentially normal CBC profiles. Furthermore, analysis of T cell distribution in the PBL revealed stable systemic activation of both CD4 and CD8 T cells in mice receiving 10D1+anti-PD-1, but not in mice receiving anti-PD-1+PP4631 or anti-PD-1+PP4637 (Figure 44), further supporting the idea that L3D10-based anti-CTLA-4 mAbs do not lead to systemic T cell activation.

[0212] Example 14. Binding characteristics of humanized anti-CTLA4 antibodies

[0213] To confirm that the humanized antibody retains its CTLA4 binding signature, we examined its binding to immobilized and plate-bound CTLA4. Figure 45 As shown, humanization does not affect binding to immobilized CTLA4, and all three humanized antibodies exhibit similar binding to the parental chimeric L3D10 antibody. However, as Figure 46 As shown, humanization further reduces the binding of L3D10 to soluble CTLA4. Based on the reduced binding to soluble CTLA4, it is expected that the three humanized antibodies will induce equivalent tumor rejection with even fewer autoimmune side effects than L3D10.

[0214] We have confirmed that the blocking activity of chimeric L3D10 is 1000 times higher than that of 10D1. This suggests an interesting possibility that blocking the B7-CTLA-4 interaction could explain its lack of irAE. Figure 47 and 48 As shown, neither PP4631 nor PP4637 blocks B7-CTL-A4 interactions in vitro or in vivo. The fact that PP4631 and PP4637 exhibit reduced irAEs further supports the idea that blocking B7-CTLA-4 interactions does not lead to an improvement in the safety of L3D10.

[0215] Given the proposed protective role of CTLA-4 against autoimmune diseases, we propose reducing binding to soluble CTLA-4 as a potential mechanism to improve the safety profile. To test this hypothesis, we used increased growth and weight gain in female mice receiving anti-PD-1 + anti-CTLA-4 mAb during the perinatal period as a baseline indicator of irAEs. Figure 42 As shown, a severe reduction in weight gain was observed in mice receiving both 10D1 and anti-PD-1, while mice receiving PP4637 + anti-PD-1 had the lowest irAE, followed by PP4631, and then L3D10. This strongly inverse correlation with reduced binding to sCTLA-4 is consistent with the central hypothesis.

[0216] Example 15. Processability assessment of humanized anti-CTLA4 antibody

[0217] To evaluate the development and manufacturing potential of three different humanized antibodies, multiple analytical methods were performed to characterize the different antibodies.

[0218]

[0219]

[0220] As a preliminary assessment, the predicted molecular weights and isoelectric points of the three leading candidate antibodies were calculated based on their amino acid sequences. As shown in Table 7, all antibodies are quite similar, but the antibody has a slightly lower PI.

[0221] Table 7: Theoretical parameters of three humanized antibodies

[0222] protein name Theoretical MW(Da) Theoretical PI PP4631 (49647.8+23483.1)X 2＝96614.0 7.9 PP4637 (49644.9+23483.1)X 2＝96611.1 7.65 PP4638 (49644.9+23311.9)X 2＝96568.7 7.9

[0223] Product yield assessment

[0224] To evaluate the production rates of different antibodies, HEK293 cells were transiently transfected with vectors expressing the heavy and light chains of different antibodies. These cells were then cultured in shake flasks for 6 days in serum-free medium. After 6 days, the supernatant was collected and the antibodies were purified by one-step protein A chromatography. As shown in Table 8 below, antibodies PP4631 and PP4637 exhibited similar protein yields, while antibody PP4638 was produced in a much lower relative yield.

[0225] Table 8: Assessment of humanized antibody production yield.

[0226] Antibody Concentration (mg / mL) OD 260 / 280 Yield (mg / L) PP4631 1.280 0.53 126 PP4637 4.532 0.53 118 PP4638 0.729 0.57 56

[0227] To assess the purity of transiently expressed antibodies, samples were analyzed by reducing and non-reducing SDS-PAGE. Figure 50As shown, samples from all three antibodies produced gel bands indicating antibody molecules, and the samples were relatively pure after purification of protein A.

[0228] Size exclusion chromatography

[0229] To further examine the purity and aggregation of different antibodies after transient expression, we performed size exclusion chromatography on the purified protein. Simply put, 50 μg of filtered (using a 0.22 μm filter) sample was used for SE-HPLC separation using a TOSOH G3000SWxl 5 μm column. PBS pH 7.4 was used as the mobile phase. As shown in Table 9 below, all humanized antibodies exhibited >90% purity after protein A purification. Antibodies PP4631 and PP4637 showed similar low levels of high molecular weight (MW) aggregation and degradation, with most of the protein in the main peak. In contrast, antibody PP4638 exhibited higher levels of aggregation and some degradation. SE-HPLC chromatograms are shown below. Figure 51 middle.

[0230] Table 9: Size Exclusion Chromatography

[0231] Antibody Gathering Main Peak degradation PP4631 2.6％ 97.4％ 0 PP4637 3.0％ 97.0％ 0 PP4638 6.5％ 92.4％ 1.1％

[0232] Capillary electrophoresis (CE)

[0233] Capillary electrophoresis is used to quantify the amount of protein within peak bands under reducing and non-reducing conditions, as well as the amount of unglycosylated heavy chain proteins. Simply put, 100 μg of sample is diluted with 2 μL of 10 kDa standard protein in CE-SDS sample buffer along with iodoacetamide (non-reducing conditions) or β-mercaptoethanol (reducing conditions). The sample is then treated at 70°C for 10 minutes. For separation, a PA-800, 50 μm ID bare fused silica capillary was used; the running length was 20.2 cm; the separation voltage was 15 kV; and the OD... 220 Used for detection. As shown in Table 10 below, all three proteins exhibited high purity levels, consistent with SDS-PAGE, and were all highly glycosylated. CE-SDS chromatograms are shown below. Figure 52 middle.

[0234] Table 10: Capillary Electrophoresis

[0235] Antibody Non-reducing % reduction% Unglycosylated heavy chains PP4631 97.3 99.5 0.3 PP4637 97.2 99.5 0.4 PP4638 96.9 99.4 0.4

[0236] Deamidation: Capillary isoelectric focusing (cIEF) and liquid chromatography-mass spectrometry (LC-MS)

[0237] The level of protein deamidation under high pH stress was determined by comparing antibodies treated with high pH stress for two different time periods (5 hours and 12.5 hours) with those not treated, followed by cIEF and LC-MS analysis.

[0238] The charge isotype distribution and isoelectric point of different antibodies were determined by capillary isoelectric focusing (cIEF). In simpler terms, the sample was buffer-exchanged to 20 mM Tris pH 8.0, and then 100 μg of sample protein was mixed with the amphoteric electrolyte methylcellulose and labeled with PI 7.05 and PI 9.77. iCE3 TM For analysis, 100μm ID capillary; 1.5kV plus 3kV; OD 280 For detection. In the deamidation stress treatment, the samples were treated with 500 mM NaHCO3 for 5 h or 12.5 h, followed by examination by cIEF and LC-MS. The results of the analysis are shown in Table 11 below and the LC-MS chromatogram is shown in Figure 53. All three antibodies showed a predicted increase in deamidation mass and a corresponding decrease in the main peak under stress conditions. As predicted by the amino acid sequence, the pI of antibody PP4637 was slightly lower than that of PP4631 and PP4638 (Table 7), and the observed pI, which was higher than the predicted pI, inferred to indicate glycosylation.

[0239] Table 11: Isoelectric focusing and deamidation

[0240]

[0241]

[0242] Differential scanning calorimetry (DSC) thermal analysis

[0243] To determine the thermal stability and melting temperature of different antibodies, they were subjected to differential scanning calorimetry (DSC) thermal analysis. Simply put, a sample of 2 mg / mL in PBS pH 7.4 was subjected to a temperature gradient from 15 °C to 105 °C at a rate of 1 °C / min. The Cp values ​​of both samples and the buffer (as background) were monitored as a function of temperature. Cp versus temperature curves were obtained after background subtraction, and the peaks indicate the Tm of the analyte. As shown in Table 12 below, all three antibodies exhibited similarly high melting temperatures. The DSC curves of the three antibodies are shown in Table 12. Figure 54 middle.

[0244] Table 12: Size Exclusion Chromatography

[0245] Antibody <![CDATA[T M (℃)]]> PP4631 75.6 PP4637 76.2 PP4638 76.6

[0246] Oxidation: Peptide Plotting

[0247] Oxidative modifications of humanized antibodies were assessed by peptide mapping using LC-MS with or without oxidative stress. Samples were denatured at 65 °C in the presence of 6 M GnCl and 5 mM β-ME, followed by acetylation with iodoacetamide. The processed samples were then digested at 55 °C with trypsin (Promega, sequencing grade), and the digested mixture was separated on a C18 reverse-phase LC column (ACQUITY UPLC BEH130 C18, 2.1 × 100 mm, 1.7 μm) and analyzed by mass spectrometry (Waters XEVO-G2S QTOF) using Masslynx and Biophatmlynx analytical tools. In oxidative stress analysis, samples were treated with 0.05% or 0.1% H2O2 for 1 h, followed by LC-MS examination. The results are shown in Tables 13-16 below.

[0248] Table 13. Oxidation of humanized antibodies at methionine sites. Top: Antibody PP4631. Middle: Antibody PP4637.

[0249] The image below shows antibody PP4638.

[0250]

[0251]

[0252]

[0253] Table 14. Oxidation of humanized antibody PP4631 at tryptophan sites. Red numbers indicate evidence of fragmentation; "—" indicates none detected; "0" indicates detected at very low levels.

[0254]

[0255] Table 15. Oxidation of humanized antibody PP4637 at tryptophan sites. Red numbers indicate evidence of fragmentation; "—" indicates none detected; "0" indicates detected at very low levels.

[0256]

[0257]

[0258] Table 16. Oxidation of humanized antibody PP4638 at tryptophan sites. Red numbers indicate evidence of fragmentation; "—" indicates none detected; "0" indicates detected at very low levels.

[0259]

[0260] Combination specificity

[0261] The binding specificity of different antibodies was determined by assessing their ability to detect non-specific binding to two different cell lines (CHO and HEK293) that do not express CTLA4 at two different concentrations relative to 10D1. Simply put, 100 μg / mL or 20 μg / mL of sample (or reference mAb) was added to PBS at 3 × 10⁻⁶ e⁻¹. 6 Cells / mL (CHO or HEK293) were incubated. FITC-labeled rabbit anti-human IgG antibodies (Boster, Wuhuan, China) were used to detect and measure the binding of the target mAbs to cells by FACS. As shown in Table 17 below, antibodies PP4631 and PP4637 exhibited very low binding and good specificity, while antibody PP4638 showed non-specific binding activity with the control cell line.

[0262] Table 17: Binding specificity with CHO and HEK293 cell lines

[0263]

[0264] Example 16. Epitope mapping of L3D10 and humanized antibody

[0265] To map the CTLA-4 binding epitopes of the parental L3D10 antibody and the humanized variants PP4631 and PP4637, we utilized the fact that mouse and human CTLA4 proteins are cross-reactive with B7-1 but not with anti-CTLA-4 antibodies. Therefore, we designed several mutants of the human CTLA-4 Fc protein, in which amino acid clusters from the human CTLA-4 protein were replaced with amino acids from the mouse CTLA-4 protein. Because the anti-CTLA-4 antibody used in this study does not bind to mouse CTLA-4, the binding of the anti-human CTLA-4 antibody should be eliminated when key residues of the antibody-binding epitope are replaced with mouse amino acids.

[0266] DNA vectors encoding 11 CTLA-4Fc mutant proteins (M1-M11) (SEQ ID NO:40-50) were constructed based on wild-type human CTLA-4Fc sequences, and the proteins were obtained by transiently transfecting them into HEK293 at a scale of 0.01 mL followed by one-step protein A chromatography purification.

[0267] The binding of anti-CTLA4 antibody to CTLA4Fc protein was performed by ELISA. Plates were coated with CTLA-4Fc protein at a concentration of 1 μg / mL, and the amount of bound protein was measured using a biotinylated antibody or B7-1Fc fusion protein in the soluble phase during the binding assay, with horseradish peroxidase (HRP) binding to streptavidin to determine the amount of bound protein.

[0268] Anti-human CTLA-4 antibodies do not cross-react with mouse CTLA-4, which presumably reflects differences in the amino acid sequences of human and mouse CTLA-4 in the extracellular domain. Figure 55 The extracellular domains of CTLA-4 in humans, macaques, and mice are compared, highlighting sequence conservation between humans and macaques, while also demonstrating numerous differences between mouse and primate sequences. Mouse and human CTLA4 proteins exhibit cross-reactivity with B7-1, attributed to conservation of the MYPPPY binding motif (72).

[0269] To map the binding epitopes of antibodies against human CTLA-4, we generated multiple non-overlapping CTLA-4 Fc mutant proteins that incorporate mouse-specific amino acid clusters into the human CTLA-4 sequence. The amino acids incorporated into each of the 11 mutants are shown in [image / description]. Figure 55 The amino acid sequences of WT and mutant CTLA-4Fc proteins are shown in Figure 56. These proteins were produced by transient transfection into HEK293 cells, and the yields are provided in Table 18. Many mutations, as indicated by their yield, affect protein expression relative to the WT human CTLA-4Fc protein.

[0270] Table 18: Transiently produced WT and mutant CTLA-4Fc proteins in HEK293 cells.

[0271] protein name Yield (mg) CTLA-4Fc WT control 0.72 Mutant 1 1.29 Mutant 2 0.03 Mutant 3 0.21 Mutant 4 0.11 Mutant 5 1.89 Mutant 6 0.38 Mutant 7 0.25 Mutant 8 1.61 Mutant 9 0.01 Mutant 10 0.04 Mutant 11 1.70

[0272] The ability of chimeric L3D10 and humanized antibodies PP4631 and PP4637 to bind immobilized CTLA-4 Fc mutant constructs was then determined by ELISA. Plates were coated with the CTLA-4 mutant constructs and biotinylated anti-CTLA-4 antibody or B7-1 Ig control protein was added. Binding was measured using HRP-conjugated streptavidin. The results of the binding analysis are shown in Tables 19-22. As expected, all four binding proteins showed good dose-dependent binding to WT CTLA-4 Fc protein. However, mutations introduced into M9 and M10 proteins altered the overall structure, and these mutants failed to bind B7-1 Fc. Mutations introduced into M2 and M4 also partially altered the CTLA-4 conformation, as indicated by reduced binding relative to WT protein. Consistent with this view, all four of these mutants (M2, M4, M9, and M10) were expressed at much lower yields (Table 18). In contrast, using binding to WT CTLA-4Fc and B7-1Fc proteins as references, M11 was clearly highlighted as a well-expressed protein, efficiently binding to B7-1Fc but failing to bind to either of the humanized anti-CTLA-4 antibodies. Its binding to the initial L3D10 was also reduced by approximately 100-fold (Table 20). As expected, the mutations affecting overall confirmation also affected binding to anti-CTLA-4 antibodies.

[0273] Table 19: Integrity of CTLA4Ig mutants as indicated by their binding to the B7-1 Ig fusion protein. Binding to CTLA4Fc protein was performed by ELISA, and the amount of biotinylated protein bound was measured by horseradish peroxidase (HRP) binding to streptavidin. Values ​​shown are OD450 measurements. WT = wild-type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins.

[0274]

[0275] Table 20: Epitope mapping of the chimeric L3D10 antibody. Binding to CTLA4Fc protein was performed by ELISA, and the amount of biotinylated protein bound was measured by horseradish peroxidase (HRP) combined with streptavidin. Values ​​shown are OD450 measurements. WT = wild-type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins.

[0276]

[0277] Table 21: Epitope mapping of humanized antibody PP4631. Binding to CTLA4Fc protein was performed by ELISA, and the amount of biotinylated protein bound was measured by horseradish peroxidase (HRP) combined with streptavidin. Values ​​shown are OD450 measurements. WT = wild-type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins.

[0278]

[0279] Table 22: Epitope mapping of humanized antibody PP4637. Binding to CTLA4Fc protein was performed by ELISA, and the amount of biotinylated protein bound was measured by horseradish peroxidase (HRP) combined with streptavidin. Values ​​shown are OD450 measurements. WT = wild-type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins.

[0280]

[0281]

[0282] Table 23: Raw data from replicate studies show a specific loss of the antigenic epitope only in M11. Similar to Tables 2-5, but additional controls are included to demonstrate the specificity of the binding.

[0283]

[0284] Because L3D10 retains significant binding to M11, we tested whether the binding was specific. We plated human CTLA4-Fc (hCTLA4Fc), mouse CTLA4-Fc (mCTLA4-Fc), control IgG1-Fc, or all mutant hCTLA4-Fc and measured their binding to B7-1Fc, as well as L3D10, PP4631, and PP4637. A large amount of data is presented in Table 23. Figure 57 As shown, biotinylated B7-1 also binds well to hCTLA-4, mCTLA-4, and M11. The specificity of the analysis is demonstrated by the lack of binding to IgG1-Fc. Interestingly, although L3D10 binds more strongly to M11 than to IgG1-Fc and mCTLA4-Fc, the significant binding to IgG1-Fc suggests that the binding of the chimeric antibody to M11 may be nonspecific. In contrast, none of the humanized antibodies bound to the controls for M11, mCTLA-4, and IgG1-Fc. These data confirm that the mutation introduced into M11 selectively eliminates the binding of L3D10, PP4631, and PP4637 to CTLA-4.

[0285] Using the known composite structure 133, we plotted the CTLA-4 epitopes in a 3D structure. For example... Figure 58 As shown, the epitopes identified by these mAbs are located within the region covered by B7-1. Therefore, the binding of L3D10, PP4631, and PP4637 to CTLA-4 will be mutually exclusive with the binding of B7-1. Poor blocking of PP4631 and PP4637 is due to lower affinity rather than a unique binding domain.

[0286] Taking advantage of the fact that mouse and human CTLA-4 proteins cross-react with B7-1, but anti-human CTLA-4 antibodies do not cross-react with mouse CTLA-4 proteins, we were able to map the binding epitopes of the L3D10-derived antibody using ELISA. Using multiple mutants of the human CTLA-4 Fc protein, with amino acid clusters derived from human CTLA-4 protein replaced by amino acid substitutions from mouse CTLA-4 protein, we clearly demonstrated that when we replaced four amino acids immediately following the known B7-1 binding domain of CTLA-4, the dose-dependent binding of the antibody was essentially eliminated. The fact that the binding epitope is located immediately adjacent to the B7-1 binding domain in the mapping is strongly correlated with the ability of the L3D10 antibody to block B7-CTLA-4 interactions in vitro and in vivo. Since soluble CTLA4 is produced by fusing the C-terminal amino acid of the extracellular IgV domain with the intracellular domain, it is easy to infer that antibodies binding to polymorphic C-terminal domain residues (only 18 amino acids from the C-terminus) are more likely to lose reactivity with soluble CTLA-4, in which the large intracellular domain is fused with the C-terminus of the extracellular domain.

[0287] To further investigate the binding domain of anti-CTLA4 antibodies, six additional mutant CTLA4-Fc fusion proteins were designed and named M12-M17 (SEQ ID NO:51-56). Figure 59 ), and used it to compare anti-CTLA4 antibody 10D1 ( Figure 60 A) PP4631 ( Figure 60 B) and PP4637 Figure 60 The combination of C). For example... Figure 60 As shown, M11 is located at position Y 103 L 104 I 106 The mutation elimination at the site of binding to 10D1, PP4631, and PP4637 confirms that the binding sites for 10D1, PP4631, and PP4637 include residue Y. 103 L 104 I 106 Importantly, the additional mutation in A29>Y is restored in Y. 103 L 104 I 106Mutated CTLA-4 binds to PP4631 and PP4637. These data confirm that position A in CTLA4... 29 The binding of antibodies PP4631 and PP4637 is important, but not for 10D1.

[0288] Example 17. Anti-CTLA-4mAb synergistically enhances tumor rejection induction with anti-4-1BB.

[0289] Studies in animal models have shown that antitumor responses induced by anti-CTLA-4 monoclonal antibodies (mAbs) are at least in part due to antigen-specific T-cell responses against normal “self” differentiation antigens (73,74). The tendency of anti-CTLA-4 antibodies to exacerbate autoimmune diseases is well-documented in mice (75-78). This idea has been further confirmed and has proven to be a major limitation in more recent human trials of patients with severe autoimmune manifestations requiring treatment interruption (79). On the other hand, cancer-therapeutic anti-4-1BB mAbs have been shown to eliminate the occurrence of autoimmune diseases in mice susceptible to lupus (24,25).

[0290] The fact that anti-4-1BB mAbs can stimulate anti-tumor responses and reduce autoimmune expression raises the intriguing possibility that combining this antibody with an anti-CTLA-4 mAb could induce cancer rejection without causing autoimmunity. In this study, the combination of anti-CTLA-4 and anti-4-1BB was used to induce rejection in large, established tumors.

[0291] Anti-mouse CTLA-4 and anti-mouse 4-1BB antibodies induce CD8 Combinations of T-cell-mediated tumor rejection effect.

[0292] Two models (one with minimal disease and one with large, established tumors) were used to test the antitumor effects of combined anti-mouse 4-1BB and anti-mouse CTLA-4mAb treatments. C57BL / 6 mice were challenged by subcutaneous inoculation with MC38 colon cancer cells, and antibodies were injected into the tumor-challenged mice at different time points after tumor cell inoculation. Tumor size and incidence were monitored by physical examination.

[0293] In the minimal disease model, starting 48 hours after tumor cell inoculation, mice were treated with hamster IgG plus rat IgG, anti-4-1BB plus hamster IgG (anti-4-1BB alone), anti-CTLA-4 plus rat IgG (anti-CTLA-4 alone), or a combination of anti-4-1BB and anti-CTLA-4. Antibodies were administered intraperitoneally (ip) on days 2, 9, and 16. Treatment with anti-4-1BB or anti-CTLA-4 mAb alone resulted in delayed tumor growth, with 1 in 5 mice in each group rejecting the tumor, while 4 out of 5 mice treated with both anti-CTLA-4 and anti-4-1BB mAb were tumor-free at the end of the experiment. Figure 61 A shows the tumor growth measurements for each mouse. To compare growth rates between groups, a linear random-effects model was applied to the data. The combination therapy significantly reduced daily tumor size growth by 4.6 mm compared to anti-CTLA-4 alone. 2 / day (p=0.0094). Furthermore, the combination therapy significantly reduced growth by 8.4 mm compared to anti-4-1BB alone. 2 / day (p = 0.0006). In addition to growth rate, actual tumor size was compared between treatment groups six weeks after initial tumor challenge. The mean tumor size at six weeks for mice given combination therapy was 27.5 mm. 2 ) and respectively treated with anti-CTLA-4 (137.8mm) 2 The tumor burden was significantly smaller in mice compared to those treated with anti-4-1BB (287.6, p = 0.0006) or anti-4-1BB (287.6, p = 0.0006). Therefore, under the minimum tumor burden setting, the combination of anti-4-1BB and anti-CTLA-4 mAb resulted in a significant delay in tumor growth compared to administration of anti-4-1BB or anti-CTLA-4, respectively.

[0294] To determine whether the antitumor effect of combined mAb treatment against small tumor burdens could be extended to therapeutic applications against larger tumor burdens, mice with established tumors were treated with antibodies. Wild-type C57BL / 6 mice were challenged by subcutaneous inoculation with MC38 colon cancer cells. After 14 days of tumor growth, mice with established tumors (typically >7 mm in diameter) were selected and randomly assigned to four treatment groups: hamster IgG plus rat IgG, anti-4-1BB plus hamster IgG, anti-CTLA-4 plus rat IgG, and a combination of anti-4-1BB mAb and anti-CTLA-4 mAb. Antibodies were administered intraperitoneally on days 14, 21, and 28 post-tumor challenge. Figure 61As shown in B, treatment with anti-CTLA-4 mAb did not inhibit tumor growth compared to control IgG treatment, but rejection was observed in one of the eight mice in the group. Treatment with anti-4-1BB mAb slightly slowed tumor growth, but only one of the eight mice rejected the tumor. In contrast, the combination therapy of both anti-CTLA-4 and anti-4-1BB mAb resulted in tumor eradication in seven of the eight mice and prevented further tumor growth in the remaining mice. As described above, growth rates between groups were compared by applying a linear random-effects model to the data. The combination therapy significantly reduced daily tumor size growth by 10.6 mm compared to anti-CTLA-4 alone. 2 / day (p<0.0001). Furthermore, the combination therapy significantly reduced growth by 6.2 mm compared to anti-4-1BB alone. 2 / day (p = 0.0002). In addition to growth rate, actual tumor size was compared between treatment groups eight weeks after initial tumor attack. Estimated mean tumor size at eight weeks for mice given combination therapy (-1.7 mm). 2 95% CI: -10.8, 7.5 mm 2 ) and respectively treated with anti-CTLA-4 (404.9mm) 2 95% CI: 285.4, 524.4 mm 2 (p<0.0001) or anti-4-1BB (228.4mm) 2 95% CI: 200.4, 689.9 mm 2 The tumor size was significantly smaller compared to mice with a tumor burden of p = 0.0004. Therefore, under a large tumor burden, the combination mAb appeared to significantly delay tumor growth as well as anti-CTLA-4 or anti-4-1BB alone.

[0295] MC38 is known to cause liver metastases. 80To evaluate the effect of therapeutic antibodies on liver metastases, liver metastases in all participating mice were analyzed histologically. As shown in Table 24, approximately 60% of the control Ig-treated mice had micrometastases in the liver. Treatment with either anti-CTLA-4 or anti-4-1BB antibody alone slightly reduced the metastasis rate, but the reduction was not statistically significant. Notably, only 1 / 22 mice in the two-antibody treatment group had liver metastases. Using a logistic regression model, we found that mice given anti-4-1BB alone had an approximately 4.7-fold higher chance of liver metastases than mice given both anti-4-1BB and anti-CTLA-4 (95% CI: 1.6, 13.7; p = 0.0050). Similarly, mice given anti-CTLA-4 alone had a 3.6-fold higher chance of liver metastases compared to mice given both treatments (95% CI: 1.3, 10.2; p = 0.0174). Therefore, combination therapy significantly reduced liver metastases of MC38 compared to treatment with either antibody alone.

[0296] Table 24: Combination therapy generally reduces liver metastasis *

[0297]

[0298] *Data are summarized from 4 independent experiments. At least two sections of each liver were examined after H&E staining.

[0299] To determine which immune cell subsets contributed to the antitumor effect induced by the combination mAb treatment, the major lymphocyte subsets were depleted with monoclonal antibodies. MC38 tumor cells were injected subcutaneously. After the tumors became palpable, the tumor-bearing mice were divided into four groups. Each group received a series of intraperitoneal antibody injections to deplete different immune cell subsets, including no depletion in the presence of normal rat IgG, CD4 T cell depletion with anti-CD4 mAb (GK 1.5), CD8 T cell depletion with anti-CD8 mAb (2.4.3), and NK cell depletion with anti-NK1.1 mAb (PK136). Additionally, all mice in all groups were treated weekly for three weeks with anti-CTLA-4 plus anti-4-1BB mAb. Adequate depletion of immune cell subsets was assessed by flow cytometry of peripheral blood taken from mice immediately prior to completion of the experiment (data not shown). As expected, mice without immune cell depletion responded to the combination treatment with anti-CTLA-4 and anti-4-1BB mAb (…). Figure 62 Similarly, depletion of NK cells and CD4 T cells did not affect the antitumor activity of the combination anti-CTLA-4 plus anti-4-1BB mAb therapy. However, depletion of CD8 T cells eliminated the antitumor activity of the combination antibody therapy. On day 28, the estimated mean tumor size in mice with depleted CD8 T cells was 92.3 mm.2 95% CI: 64.5, 120.1 mm 2 The size was significantly larger than that of mice without immune cell depletion (28.7 mm). 2 95% CI: -17.1, 74.4 mm 2 Mice with depleted CD4 T cells (16.7 mm) 2 95% CI: 1.0, 32.4 mm 2 ) and mice with depleted NK cells (9.3 mm) 2 95% CI: -8.3, 26.9 mm 2 The average tumor size was [data missing]. These data confirm that the tumor eradication effect of anti-CTLA-4 and anti-4-1BBmAb treatment is CD8 T cell dependent.

[0300] Anti-4-1BB antibodies reduce antibody responses to xenogeneic anti-CTLA-4 antibodies.

[0301] One of the obstacles to repeated antibody therapy is the enhancement of the host antibody response to the therapeutic antibody. 81 Because 4-1BB is known to reduce antibody responses to proteins, we evaluated the role of anti-4-1BB antibodies in the host response to CTLA-4 antibodies. Figure 63 As shown, very little (if present) anti-antibody response was detected in mice treated with control IgG or anti-4-1BB. This contrasts with the ability of anti-CTLA-4mAb to promote CD4 T cell responses. 82 Consistent with this, mice treated with anti-CTLA-4 plus rat IgG showed a strong host antibody response to the administered 4F10 antibody and rat IgG. Figure 63 (AB). When anti-4-1BB was co-administered with anti-CTLA-4mAb, this response was reduced by more than 30-fold. These data suggest that anti-4-1BB antibodies may increase the duration of other co-administered therapeutic proteins by reducing the host response to therapeutic agents.

[0302] In human CTLA-4 knock-in mice, the combination of anti-mouse 4-1BB and anti-human CTLA-4 antibodies induced tumor resection. Repellent and persistent cancer immunity.

[0303] Because anti-4-1BB reduces the production of antibodies against anti-CTLA-4, an interesting question arises: is the enhanced tumor rejection induced by anti-4-1BB solely attributable to its role in suppressing the antibody response? This human CTLA4 knock-in mouse model allows us to test whether the anti-tumor effect of anti-human CTLA4 antibodies can be enhanced by anti-4-1BB antibodies. Figure 64As shown in Figure A, while both anti-human CTLA-4 (L3D10) and anti-4-1BB antibody (2A) alone resulted in delayed tumor growth, the combination of the two antibodies led to the most significant tumor rejection. In the groups treated with anti-CTLA-4, 4-1BB, or both antibodies, 1 / 7, 2 / 7, and 5 / 7 mice, respectively, never developed tumors, while all mice in the untreated group developed tumors. Because the anti-human CTLA-4 antibody is mouse-derived, the effect of the 4-1BB antibody cannot be attributed to its inhibitory effect on the treatment of the anti-CTLA-4 antibody. Furthermore, our data also confirm that the superior effects of combination therapy may be applicable to immunotherapies based on anti-human CTLA-4 antibodies.

[0304] To test whether mice treated with dual antibodies were immune to further tumor cell attack, we attacked them with tumor cells 110 days after their first tumor cell attack. Figure 64 As shown in Figure B, all five mice treated with the dual antibodies, which had already rejected tumor cells in the first round, remained tumor-free, while the untreated control mice exhibited progressive tumor growth. Therefore, the combination therapy also induced durable immunity against cancer cells.

[0305] One of the obstacles to protein-based immunotherapy is host immunity to the therapeutic protein. In the case of antibodies, the host can install antibodies onto xenotype, allotype, and individual genotype epitopes. 81 Xenotype responses can be eliminated through complete humanization, but other anti-antibody responses require special consideration. Impairment is more pronounced with anti-CTLA-4 antibodies because they are adjuvants. Previous studies by Mittler et al. demonstrated significant suppression of T-cell-dependent humoral immune responses. 83 Our data confirm that co-administration of anti-4-1BB antibodies reduces the host response to anti-CTLA-4 antibodies, suggesting another advantage of combination therapy using anti-CTLA-4 and anti-4-1BB antibodies.

[0306] In summary, our data confirm that the combination therapy of anti-CTLA-4 and anti-4-1BB antibodies provides three main benefits: increased cancer immune effects, mutual suppression of autoimmune side effects, and improved anti-antibody responses.

[0307] All disclosures and patents mentioned in this specification are incorporated herein by reference as if each individual disclosure or patent application were specifically and individually indicated to be incorporated in its entirety. While the invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications are possible, and this application is intended to cover the invention generally in accordance with the principles of the invention and to include any variations, uses, or adaptations that deviate from this disclosure if such deviations occur within the known or customary practice in the field to which the invention pertains and are applicable to the essential features set forth above.

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Claims

1. An anti-CTLA4 antibody comprising (a) a light chain variable region comprising (i) a complementarity-determining region (CDR1) wherein the amino acid sequence of the CDR1 region is shown in SEQ ID NO: 21, (ii) a CDR2 region wherein the amino acid sequence of the CDR2 region is shown in SEQ ID NO: 37, and (iii) a CDR3 region wherein the amino acid sequence of the CDR3 region is shown in SEQ ID NO: 23; and (b) a heavy chain variable region comprising (i) a CDR1 region wherein the amino acid sequence of the CDR1 region is shown in SEQ ID NO: 24, (ii) a CDR2 region wherein the amino acid sequence of the CDR2 region is shown in SEQ ID NO: 35, and (iii) a CDR3 region wherein the amino acid sequence of the CDR3 region is shown in SEQ ID NO:

26.

2. The anti-CTLA4 antibody according to claim 1, comprising: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 64; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

71.

3. The antigen-binding fragment of the anti-CTLA4 antibody according to claim 1 or 2.

4. The antigen-binding fragment according to claim 3, wherein the fragment is selected from the group consisting of Fab', F(ab')2, Fv and ScFv.

5. A pharmaceutical composition comprising a therapeutically effective amount of the anti-CTLA4 antibody according to claim 1 or 2 or the antigen-binding fragment according to claim 3 or 4, and a physiologically acceptable carrier or excipient.

6. Use of the anti-CTLA4 antibody according to claim 1 or 2, or the antigen-binding fragment according to claim 3 or 4, or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating cancer in a subject.

7. The use according to claim 6, wherein the cancer is selected from the group consisting of: carcinoma; hematopoietic tumors of the lymphatic lineage; leukemia; hematopoietic tumors of the bone marrow lineage; mesenchymal tumors; and tumors of the central and peripheral nervous systems.

8. The use according to claim 7, wherein the cancer is selected from the group consisting of: B-cell lymphoma; T-cell lymphoma; Burkitt lymphoma; fibrosarcoma; rhabdomyosarcoma; melanoma; seminoma; neuroblastoma; glioma; astrocytoma; schwannoma; osteosarcoma; xeroderma pigmentosum; and follicular thyroid carcinoma.

9. The use according to claim 7, wherein the cancer is an epithelial carcinoma, and wherein the epithelial carcinoma is bladder epithelial carcinoma; breast epithelial carcinoma; colon epithelial carcinoma; renal epithelial carcinoma; hepatic epithelial carcinoma; lung epithelial carcinoma; ovarian epithelial carcinoma; pancreatic epithelial carcinoma; gastric epithelial carcinoma; cervical epithelial carcinoma; thyroid epithelial carcinoma; skin epithelial carcinoma; squamous cell carcinoma; teratoma; hepatocellular carcinoma; or prostate epithelial carcinoma.

10. The use according to claim 9, wherein the epithelial carcinoma is colonic epithelial carcinoma.

11. The use according to claim 7, wherein the cancer is leukemia, and wherein the leukemia is acute lymphoblastic leukemia; acute or chronic myelogenous leukemia; or chronic lymphoblastic leukemia.

12. The use according to claim 6, wherein the cancer is selected from the group consisting of: ovarian cancer, bladder cancer, colon cancer, skin cancer, pancreatic cancer, uterine cancer, sarcoma, melanoma, lung cancer, breast cancer, and Hodgkin's or non-Hodgkin's lymphoma.

13. The use according to claim 12, wherein the cancer is melanoma.

14. The use according to any one of claims 6-13, wherein the anti-CTLA4 antibody is combined with another pharmaceutical agent selected from the group consisting of anti-PD-1 and anti-4-1BB antibodies.

15. The use according to claim 14, wherein the anti-PD-1 or anti-4-1BB antibody and the anti-CTLA4 antibody are combined in a single molecule as bispecific antibodies.

16. The use according to claim 6, wherein the drug induces strong Treg loss and local T cell activation in the tumor microenvironment, but the induced systemic T cell activation is minimal.

17. The use according to claim 12, wherein the cancer is lung cancer.

18. The use according to claim 12, wherein the cancer is ovarian cancer.

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