Chimeric and humanized Anti-human CTLA4 monoclonal antibodies and uses thereof

Abstract

To provide compositions of chimeric and humanized antibodies that bind to the human CTLA4 molecule and their use in cancer immunotherapy and for reduction of autoimmune side effects compared to other immunotherapeutic agents.SOLUTION: The invention relates to enhanced CTLA4 blocking activity for CTLA4 ligands B7.1 and B7.2, enhanced effector function, or reduced binding to soluble CTLA4 relative to membrane bound or immobilized CTLA4. An 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 set forth in SEQ ID NO: 1, and a heavy chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to chimeric and humanized antibodies that bind to the human CTLA4 molecule and methods of their use. [Background technology]

[0002] The immune systems of humans and other animals serve to provide protection against infection and disease. Such 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 cells (NK), and antigen-specific cytotoxic T lymphocytes by T cells, and the release of various cytokines in response to antigen recognition.

[0003] The ability of T cells to optimally mediate immune responses to antigens requires two distinct signaling interactions. First, antigens arranged on the surface of antigen-presenting cells (APCs) must be presented to antigen-specific naive T cells in the form of MHC-peptide complexes (1, 2). Such presentation delivers a signal via the T cell receptor (TCR) that instructs the T cell to mount an immune response specific to the presented antigen. Second, a series of costimulatory signals mediated through interactions between APCs and distinct T cell surface molecules initially induces T cell activation and proliferation and ultimately their inhibition (3-5). Thus, the first signal confers specificity to the immune response, while the second signal serves to determine the nature, strength, and duration of the response while limiting immunity to self. Particularly important among these second signaling molecules is the binding between the B7.1 (CD80) ( 6 ) and B7.2 (CD86) ( 7 – 9 ) ligands on antigen-presenting cells and the CD28 and CTLA4 receptors on T lymphocytes ( 10 – 12 ).

[0004] Cytotoxic T-lymphocyte antigen 4 (CTLA4) is recognized as a key regulator of adaptive immune responses, playing a central role in maintaining peripheral immune tolerance and shaping the repertoire of emergency T cell responses, and is therefore a therapeutic target for the treatment of cancer and inflammation. Treatment with anti-CTLA4 antibodies has been shown to be a powerful tool for enhancing antitumor immunity in preclinical models (10). Monotherapy with antibodies against CTLA4 promoted the rejection of transplantable tumors of various origins.

[0005] Based on promising preclinical tumor model studies, the clinical potential of antibodies against CTLA4 has been explored in different human malignancies. Anti-CTLA4 (ipilimumab, marketed as Yervoy) has shown efficacy in the treatment of melanoma, but therapeutic targeting of CTLA4 has been associated with autoimmune-like toxicities. The characteristic side effects of CTLA4 inhibition are commonly referred to as immune-related adverse events (irAEs), with the most common irAEs being rash, hepatitis, colitis, and endocrine disorders, particularly hypopituitarism. Therefore, it is desirable to improve the therapeutic potential of anti-CTLA4 antibodies by increasing efficacy while reducing associated irAEs.

[0006] Another focus in the field of immunotherapy and tumor treatment is the combination of different immune checkpoint inhibitors to enhance antitumor 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 regulate cancer immunity without enhancing autoimmunity.

[0007] Further research into the ligands of the CD28 receptor led to the identification and characterization of a series of related B7 molecules (the "B7 superfamily") (32-33). Currently, there are several known members of this family: B7.1 (CD80), B7.2 (CD86), inducible costimulatory 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).

[0008] B7-H1 is widely expressed in different human and mouse tissues, including the heart, placenta, muscle, fetal liver, spleen, lymph nodes, and thymus in both species, and in the liver, lung, and kidney of mice only (37). B7-H1 (PD-L1, CD274) is a particularly important member of the B7 superfamily due to its crucial role in shaping the immune response to tumors (38; U.S. Patent Nos. 6,803,192 and 7,794,710; U.S. Patent Application Publication Nos. 2005 / 0059051, 2009 / 0055944, 2009 / 0274666, and 2009 / 0313687; International Publication Nos. WO01 / 39722 and WO02 / 086083).

[0009] Programmed death-1 ("PD-1") is a receptor for B7-H1 and B7-DC. PD-1 is a type I membrane protein member of the extended CD28 / CTLA4 family of T cell regulators (39; U.S. Patent Application Publication Nos. 2007 / 0202100, 2008 / 0311117, 2009 / 00110667; U.S. Patent Nos. 6,808,710, 7,101,550, 7,488,802, 7,635,757, 7,722,868; International Publication No. WO 01 / 14557). Compared to CTLA4, PD-1 negatively regulates immune responses more broadly. PD-1 is expressed on activated T cells, B cells, and monocytes ( 40 – 41 ) and at low levels on natural killer (NK) T cells ( 42 – 43 ).

[0010] The interaction of B7-H1 with PD-1 has been shown to provide an important negative costimulatory signal for T and B cells (43) and function as a cell death inducer (39). The role of B7-H1 and PD-1 in inhibiting T cell activation and proliferation suggests that these biomolecules may serve as therapeutic targets for the treatment of inflammation and cancer. Therefore, the use of anti-PD1 and anti-B7-H1 antibodies to treat infections and tumors and to upregulate adaptive immune responses has been proposed and has been demonstrated to be effective in treating a number of human tumors. However, because not all patients have responded to or achieved complete remission with anti-PD-1 or anti-B7-H1 treatment, there is strong interest in combining anti-PD-1 or anti-B7-H1 antibodies with other immune checkpoint inhibitors to enhance anti-tumor activity.

[0011] 4-1BB (also known as CD137 and TNFRSF9) is another immune checkpoint molecule. The best-characterized activity of CD137 is its costimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival activity, and cytolytic activity. Furthermore, similar to anti-CTLA4, anti-4-1BB antibodies can enhance immune activity and eliminate tumors in mice (27-29). However, unlike the tendency of anti-CTLA4 antibodies to exacerbate autoimmune disease, anti-4-1BB mAbs, a cancer therapeutic, have been shown to suppress the development of autoimmune disease in lupus-affected mice, suppressing anti-dsDNA antibody production and reducing renal pathology (25, 26). Previous data have demonstrated that combining anti-CTLA4 and anti-4-1BB antibody treatments can reduce the autoimmune side effects of anti-CTLA4 treatment in a mouse colon cancer tumor model while enhancing antitumor activity (19). This indicates that it is possible to counteract the autoimmune side effects of anti-CTLA4 tumor therapy.

[0012] Preclinical screening of anti-human CTLA4 antibodies is challenging because in vitro immunological correlates can be of little value, as demonstrated by experiments with anti-mouse CTLA4 antibodies. The same anti-mouse CTLA4 antibodies that induce potent antitumor immunity in vivo can have varying effects on T cells in vitro. Anti-CTLA4 antibodies enhanced T cell proliferation in response to alloantigen but suppressed T cell proliferation in response to anti-CD28 costimulation (30, 31). Furthermore, CTLA4 binding by antibodies can promote or inhibit the proliferation of different subsets of T cells in the same culture (32). This complexity can be overcome by studying human T cell responses in rodent models.

[0013] Described herein are anti-CTLA4 antibodies for use in anti-tumor therapy that have reduced autoimmune side effects when used to enhance immune responses. Furthermore, these antibodies can be used in combination with other checkpoint inhibitors, such as anti-PD-1 and anti-4-1BB, to enhance anti-tumor efficacy while suppressing autoimmune side effects. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 6,803,192 [Patent Document 2] U.S. Patent No. 7,794,710 [Patent Document 3] US Patent Application Publication No. 2005 / 0059051 [Patent Document 4] US Patent Application Publication No. 2009 / 0055944 [Patent Document 5] US Patent Application Publication No. 2009 / 0274666 [Patent Document 6] US Patent Application Publication No. 2009 / 0313687 [Patent Document 7] WO 01 / 39722 [Patent Document 8] International Publication No. 02 / 086083 [Patent Document 9] U.S. Patent Application Publication No. 2007 / 0202100 [Patent Document 10] US Patent Application Publication No. 2008 / 0311117 [Patent Document 11] US Patent Application Publication No. 2009 / 00110667 [Patent Document 12] U.S. Patent No. 6,808,710 [Patent Document 13] U.S. Patent No. 7,101,550 [Patent Document 14] U.S. Patent No. 7,488,802 [Patent Document 15] U.S. Patent No. 7,635,757 [Patent Document 16] U.S. Patent No. 7,722,868 [Patent Document 17] WO 01 / 14557 Summary of the Invention [Means for solving the problem]

[0015] The present invention relates to antibody compositions and antigen-binding fragments thereof that bind to the human CTLA4 molecule and their use for cancer immunotherapy with reduced autoimmune side effects. Specifically, the present invention relates to enhanced CTLA4 blocking activity, enhanced effector function, or reduced binding of soluble CTLA4 to membrane-bound or immobilized CTLA4 against the 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 the light chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1, and a heavy chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 2. The antibody may also comprise a heavy chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 27, 28, or 29, and a light chain variable amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 30, 31, or 32. The antibody may comprise a light chain variable region having the CDR sequences set forth in SEQ ID NOs: 21, 22, and 23, and a heavy chain variable region having the CDR sequences set forth in SEQ ID NOs: 24, 25, and 26. More specifically, the antibody may comprise a heavy chain variable region having the CDR2 sequence set forth in SEQ ID NO: 33, 34, or 35, and a light chain variable region having the CDR sequences set forth in SEQ ID NO: 36, 37, or 38.

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

[0018] Further provided herein are antigen-binding fragments of the antibodies described herein. Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the antibodies described herein. The pharmaceutical composition may include a physiologically acceptable carrier or excipient.

[0019] In another aspect, provided herein are methods for enhancing one or more immune functions or responses in a subject, comprising administering to a subject in need thereof an anti-CTLA4 antibody composition or pharmaceutical composition described herein. In certain embodiments, provided herein are methods for preventing, treating, and / or managing diseases in which it is desirable to activate or enhance one or more immune functions or responses. The disease may be cancer, which may be a human malignancy. Specifically, the human malignancy may be melanoma, lung cancer, breast cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, Hodgkin's lymphoma or non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, or renal cell carcinoma. In another embodiment, the disease being treated is an infectious disease. The methods described herein can minimize autoimmune adverse effects associated with immunotherapy.

[0020] In other specific embodiments, the method includes a combination therapy in which the anti-CTLA4 antibody composition described herein is administered to a subject in combination with another treatment that can activate or enhance one or more immune functions or immune responses. In another embodiment, the anti-CTLA4 antibody composition described herein is administered as an adjuvant in combination with an antigenic composition. In a specific embodiment, the anti-CTLA4 antibody composition described herein is administered in combination with a vaccine composition to activate or enhance the immune response elicited by the vaccine composition.

[0021] In certain embodiments, the anti-CTLA4 antibody compositions described herein are administered to a subject in combination with one or more other therapies targeting different immunoregulatory pathways. In preferred embodiments, the activity of the therapies targeting different immunoregulatory pathways is complementary or synergistic with the anti-CTLA4 antibody compositions described herein. In some cases, the anti-CTLA4 antibody compositions described herein are administered in combination with other checkpoint inhibitors or small cancer immunomodulators, such as indoleamine-2,3-dioxygenase (IDO) inhibitors. In other cases, the anti-CTLA4 antibody compositions described herein are administered in combination with immune stimulators. Certain embodiments include combining an 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, an anti-CTLA4 antibody composition described herein and a second immune stimulatory agent are combined in a single bispecific antibody.

[0022] In another embodiment, the anti-human CTLA4 antibodies described herein can preferentially bind to human CTLA-4 expressed on the cell surface relative to soluble CTLA4 molecules. The anti-human CTLA4 antibodies can bind to human CTLA4 and preferentially upregulate the in vivo expression of B7.1 or B7.2. The antibodies can be included in compositions for use in modulating immune responses (immunotherapy) and treating cancer.

[0023] The present invention further relates to a method for screening anti-human CTLA4 mAbs with desirable activity. Preclinical screening of anti-human CTLA4 mAbs is challenging because in vitro immunological correlates of cancer immunity and autoimmune adverse effects have not been defined. Significant autoimmune side effects have been observed in clinical trials using human anti-CTLA4 (ipilimumab), particularly when combined with anti-PD-1. To identify anti-CTLA4 antibodies with reduced immune-related toxicity, human CTLA4 gene knock-in mice can be used to screen antibodies that exhibit anti-tumor activity in humanized mice for their ability to reduce autoimmune adverse effects in vivo.

[0024] In another embodiment, the present invention relates to a method for screening for anti-human CTLA4 mAbs with enhanced anti-tumor effects, wherein the antibodies exhibit enhanced local depletion of Treg cells in the tumor environment.

[0025] In yet 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 present invention further contemplates biomarkers for measuring the in vivo biological activity of anti-CTLA4 antibodies and for monitoring the apparent response to anti-CTLA4 treatment by measuring the expression levels of B7.1 and B7.2 on immune cells ex vivo.

[0026] To map the CTLA4-binding epitopes of the L3D10 parent antibody and the humanized variants PP4631 and PP4637, we took advantage of the fact that mouse and human CTLA4 proteins are cross-reactive with B7-1 but not with anti-CTLA-4 antibodies. Therefore, we designed a number of mutants of the human CTLA-4Fc protein in which clusters of amino acids 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, substituting key residues of the antibody-binding epitope with mouse amino acids can abolish binding of the anti-human CTLA-4 antibody. [Brief explanation of the drawings]

[0027] [Figure 1] Schematic representation of the chimeric (left) and humanized (right) L3D10 antibodies with novel combinations of mutations in the IgG1 Fc region. The positions of mutations within the Fc region are identified by the number at their amino acid position, and amino acids are identified by their single-letter code: the letter before the number represents the replaced amino acid, and the letter after the number represents the introduced amino acid. The variable regions of the antibody are shown as open ovals, and the human sequences are shown as gray rectangles. V = variable region, C = constant region, L = light chain, H = heavy chain. [Figure 2] CTLA4 binding of chimeric L3D10 and 10D1 to plate-immobilized CTLA4 determined by ELISA. ELISA plates were coated with 1 μg / ml of CTLA4-His protein (Sino Biological, China). Biotinylated binding proteins were added at a given concentration, and binding was measured using HRP-conjugated streptavidin. 10D1-1 and 10D1-2 are two independent lots of the same antibody. B7.1-Fc is a positive control, and Fc is a negative control. [Figure 3]L3D10 competition assay. 10D1 is less efficient at blocking chimeric L3D10 binding to CTLA4 than chimeric L3D10. The experiment was performed as in Figure 2, except that biotinylated chimeric L3D10 was mixed with a given concentration of unlabeled CTLA4-binding protein or CTLA4-Fc before addition to the ELISA plate. Note the much better blocking by unlabeled L3D10 than 10D1, suggesting that the antibody binding sites are not identical. [Figure 4] Blocking of CTLA4 binding to plate-immobilized B7.1. B7.1Fc protein was coated onto ELISA plates at 0.5 μg / ml. After washing and blocking, biotinylated CTLA4-Fc protein was added at 0.25 μg / ml in the presence of the given concentrations of competitor protein. Data shown are the mean optical density values ​​measured in duplicate at 405 nM. B7.1-Fc, chimeric L3D10, and CTLA4-Fc all blocked the CTLA4:B7.1 interaction in a dose-dependent manner, whereas two separate lots of 10D1 antibody failed to block at all doses tested. Biotinylation of CTLA4 does not disrupt the 10D1 epitope on CTLA4, as both lots of 10D1 showed strong binding to biotinylated CTLA4 (data not shown). [Figure 5] Blocking of CTLA4 binding to plate-immobilized B7.2. B7.2 Fc protein was coated onto ELISA plates at 0.5 μg / ml. After washing and blocking, biotinylated CTLA4-Fc protein was added at 0.25 μg / ml in the presence of a given concentration of competitor protein. Chimeric L3D10 blocked the CTLA4:B7.2 interaction in a dose-dependent manner, whereas two separate lots of 10D1 antibody failed to completely block the CTLA4:B7.2 interaction, even at the highest concentration. [Figure 6]Using soluble B7-1 and B7-2 and immobilized CTLA4-Fc, both 10D1 and L3D10 potently blocked the B7-CTLA4 interaction. Various doses of anti-human CTLA4 mAb were added to a plate coated with human B7-1Fc along with 0.25 μg / ml biotinylated human CTLA4-Fc. The amount of plate-bound CTLA4 was measured using HRP-conjugated streptavidin. Data shown are the average of duplicate determinations and are representative of two independent experiments. [Figure 7] Blocking CTLA4 binding to cell surface-expressed B7.1. Biotinylated CTLA4-Fc protein was added at 0.5 μg / ml to B7.1-expressing CHO cells in the presence of a given concentration of competitor protein. Binding of biotinylated fusion proteins to CHO cells transfected with mouse or human B7-1 and B7-2 was determined by flow cytometry. The amount of bound receptor was measured using phycoerythrin-conjugated streptavidin. Data shown are the average fluorescence intensity of triplicate samples. Chimeric L3D10 blocked the CTLA4:B7.1 interaction in a dose-dependent manner, whereas two separate lots of 10D1 antibody failed to block at all doses tested. [Figure 8] Blocks CTLA4 binding to cell surface-expressed murine B7.1. 10D1 shows slight but detectable blocking of murine B7-1-human CTLA4 interaction when mB7-1 is expressed on CHO cells. Various doses of anti-human CTLA4 mAb were added to CHO cells expressing murine B7-1 together with 0.25 μg / ml human CTLA4-Fc. Data shown are mean and SEM or triplicate data and are representative of two independent experiments. [Figure 9]Blocks CTLA4 binding to cell surface-expressed B7.2. Biotinylated CTLA4-Fc protein was added at 0.5 μg / ml to B7.2-expressing CHO cells in the presence of a given concentration of competitor protein. Chimeric L3D10 blocked the CTLA4:B7.2 interaction in a dose-dependent manner, whereas two separate lots of 10D1 antibody failed to completely block the CTLA4:B7.2 interaction, even at the highest concentration. Data shown in this figure were repeated at least five times. [Figure 10] 10D1 binds biotinylated human CTLA4-Fc better than L3D10. Various doses of anti-human CTLA4 mAb or control IgG were coated onto plates. Biotinylated CTLA4-Fc was added at 0.25 μg / ml. The amount of plate-bound CTLA4 was measured using HRP-conjugated streptavidin. Data shown are the average of duplicate determinations and are representative of two independent experiments. [Figure 11] L3D10, but not 10D1, blocks the interaction between polyhistidine-tagged CTLA4 and CHO cells expressing human B7-1. CHO cells expressing human B7-1 were incubated with polyhistidine-tagged CTLA4 along with a given dose of antibody, and the amount of CTLA4-Fc was detected using PE-streptavidin and measured by FACSCanto II. Data shown are the average fluorescence intensity of triplicate samples and are representative of two independent experiments. [Figure 12] Chimeric L3D10 induces complete remission of established tumors in the syngeneic MC38 model. The upper panel shows the experimental design, and the lower panel shows the growth kinetics of MC38 tumors in mice administered either control IgG (lower left panel, n=6) or chimeric L3D10 (lower right panel, n=5). [Figure 13]Therapeutic effects of chimeric L3D10 and 10D1 in the MC38 tumor model. Human CTLA4-knockin mice weighing approximately 20 grams were used in the study. 1 × 10 MC38 tumor cells were subcutaneously injected into CTLA4 h / h mice. When tumors reached a size of 0.5 cm in diameter, tumor-bearing mice were randomized into three groups containing 5 or 6 mice each. Mice were then treated (intraperitoneally) with 100 μg / injection of 10D1, chimeric L3D10, or control hIgGFc on days 7, 10, 13, and 16, as indicated by the arrows. Results from two experiments are shown (left and right panels), and data shown are the mean and standard deviation of tumor size (left panel: n = 6 / group, right panel: n = 5 / group). L3D10 and 10D1 have similar therapeutic effects in this model, and both are capable of inducing complete remission of established tumors. Tumor diameter (d) was calculated using the following formula: D = √(ab), V = ab2 / 2 (a is the major axis and b is the minor axis). Statistical analysis was performed by two-way repeated measures ANOVA (treatment x time). Left panel: P = 10D1 vs. hIgGFc: 5.71e-07; L3D10 vs. hIgGFc: P = 5.53e-07; 10D1 vs. L3D10: P = 0.869. [Figure 14] Effective rejection of MC38 by anti-CTLA-4 mAb in CTLA4h / m mice. Same as Figure 13, except heterozygous CTLA4h / m mice were used. Data shown are mean and SEM of tumor diameter (6 mice / group); 10D1 vs. hIgGFc: P=0.0011; L3D10 vs. hIgGFc: P=5.55e-05; 10D1 vs. L3D10: P=0.0346. [Figure 15] Therapeutic effects of chimeric L3D10 and 10D1 in the B16-F1 melanoma tumor model. Human CTLA4-knockin mice weighing approximately 20 grams were used in the study. Arrows indicate the time of treatment (50 μg / mouse / treatment). Data shown are the mean and standard deviation of tumor size (n=4 / group). L3D10 had similar therapeutic effects in this model, and both were able to delay tumor growth in this aggressive and poorly immunogenic tumor model. [Figure 16]Assay for measuring CTLA4 blockade in vivo. B7.1 or B7.2 binding on dendritic cells binds to and is downregulated by CTLA4 on the surface of T cells. However, binding of a blocking anti-CTLA4 antibody prevents B7.1 / B7.2 from binding to CTLA4, thus preventing downregulation of B7.1 and B7.2, resulting in a net increase in B7.1 / B7.2 expression. However, when chimeric T cells expressing both human and mouse CTLA4 are used, antibodies that bind to human CTLA4 do not prevent B7.1 / B7.2 from binding to mouse CTLA4, and the inhibition of B7.1 / B7.2 is restored. [Figure 17]10D1 does not block B7-CTLA4 interaction in vivo. Using the assay described in Figure 11, we assayed B7.1 and B7.2 expression using cells from mice treated with anti-CTLA4 antibodies. Figure 17A shows a diagram of the experimental design. Briefly, age- and sex-matched mice were intraperitoneally administered 500 μg of antibodies or their controls. Twenty-four hours after injection, the mice were sacrificed, and their splenocytes were stained with anti-CD11c, CD11b, anti-B7-1, and anti-B7-2 mAbs. Figure 17B shows representative data demonstrating the phenotype of CD11chi DCs analyzed for B7 expression. Figure 17C shows representative histograms showing the levels of B7-1 on DCs from mice administered control IgG1-Fc, L3D10, or 10D1. The data in the upper panel show antibody effects in homozygous knock-in mice, and the data in the lower panel show antibody effects in heterozygous mice. Figure 17D is similar to Figure 17C, except that B7-2 expression is shown. Data shown in Figures 17C and 17D are representative of data from three mice per group and were repeated once with three mice per group. Figure 17E shows that L3D10, but not 10D1, induced the expression of B7-1 (left panel) and B7-2 (right panel) in human CTLA4 homozygous mice. The data shown are summarized from two experiments involving a total of six mice per group. In each experiment, the average data of control mice was experimentally defined as 100%, and experimental group data were normalized to the control. Figure 17F is similar to Figure 17E, except that heterozygous mice were used. Neither L3D10 nor 10D1 blocked the B7-CTLA4 interaction in mice codominantly expressing both the mouse and human Ctla4 genes. [Figure 18] L3D10 binds to human CTLA4 but not mouse CTLA4. Data shown are dot plots of intracellular staining of CTLA4 among gated Cd3+Cd4+ cells using splenocytes from Ctla4h / h (top) or Ctla4m / m (bottom) mice. Anti-mouse CTLA4 mAb 4F10 was used as a control. [Figure 19]Therapeutic effects of chimeric L3D10 and 10D1 in CTLA4h / m mice. The upper panel shows the experimental design. CTLA4h / h mice were administered the colon cancer cell line MC38. When tumors reached a size of 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 lower panel shows the growth kinetics of MC38 tumors in mice administered either control IgG, chimeric L3D10, or 10D1 (n=6 / group). Despite the clear difference in in vivo CTLA4 blocking activity as shown in Figure 16, both L3D10 and 10D1 exhibit potent antitumor activity against the MC38 model in chimeric CTLA4m / h mice. [Figure 20] 10D1 and L3D10 have similar therapeutic effects on B16 melanoma growth. 1 × 10 B16 tumor cells were injected (subcutaneously) into Ctla4h / h mice (n = 4-5) and treated (intraperitoneally) with 100 μg (FIG. 20A) or 250 μg (FIG. 20B) of 10D1, L3D10, or control IgGFc on days 11, 14, and 17 (FIG. 20A), or days 2, 5, and 8 (FIG. 20B), as indicated by the arrows. In FIG. 20A, 10D1 vs. hIgGFc: P = 0.0265; L3D10 vs. hIgGFc: P = 0.0487; 10D1 vs. L3D10: P = 0.302. In Figure 20B, 10D1 vs. hIgGFc: P = 0.00616; L3D10 vs. hIgGFc: P = 0.0269; 10D1 vs. L3D10: P = 0.370. Data represent the mean ± SEM of 4-5 mice per group. Statistical analysis was performed by two-way repeated measures ANOVA. [Figure 21] Immunotherapeutic efficacy between L3D10 and 10D1 in Ctla4h / h (Fig. 21A) and Ctla4m / h (Fig. 21B) mice sacrificed before complete rejection to assess Treg depletion within the tumor microenvironment. Data shown are mean and SEM of tumor diameters from two independent experiments with 5 mice per group. [Figure 22]Blockade of B7-CTLA4 interaction does not contribute to the cancer immunotherapy activity of anti-CTLA4 mAbs. Figure 22A shows comparable immunotherapeutic efficacy despite the significantly different blocking activities of the two anti-CTLA4 mAbs. 5 × 10 MC38 tumor cells were injected (subcutaneously) into Ctla4 h / h mice (n = 6) and treated (intraperitoneally) with 100 μg of 10D1, L3D10, or control hIgG-Fc on days 7, 10, 13, and 16, as indicated by the arrows. Data represent the mean ± SEM of six mice per group. Statistical analysis was performed by two-way repeated-measures ANOVA (treatment × time). 10D1 vs. hIgG-Fc: P = 5.71e-07; L3D10 vs. hIgG-Fc: P = 5.53e-07; 10D1 vs. L3D10: P = 0.869. Data are representative of three independent experiments. Figure 22B: Neither antibody blocks the B7-CTLA4 interaction and induces potent tumor rejection in mice. Similar to Figure 22A, except heterozygous mice expressing both murine and human CTLA4 were used. 10D1 vs. hIgG-Fc: P = 0.0011; L3D10 vs. hIgG-Fc: P = 5.55e-05; 10D1 vs. L3D10: P = 0.0346. Data are representative of three independent experiments. Figures 22C-F: Blockade of the B7-CTLA4 interaction does not contribute to the selective depletion of Tregs in the tumor microenvironment. Figures 22C and D: Despite their ability to block the B7-CTLA4 interaction, L3D10 and 10D1 do not deplete Tregs in the spleen. Data shown are the % of Foxp3+ cells among splenic CD4 T cells in Ctla4h / h (Figure 22C) and Ctla4m / h (Figure 22D) mice. n=6. e and f: Both L3D10 and 10D1 lack Tregs among tumor-infiltrating CD4 T cells in Ctla4h / h (Figure 22E) and Ctla4m / h (Figure 22F) mice. Data shown in c–f are the % of Tregs 17 (Experiment 1) or 19 (Experiment 2) days after tumor cell challenge and 10 or 12 days after initiation of treatment with the four anti-CTLA4 mAbs, as indicated by the arrows. [Figure 23]Evaluation of the blocking activity of the commonly used anti-mouse CTLA4 mAbs 9H10 and 9D9. Figures 23A and 23B show that 9H10 does not block the B7-CTLA4 interaction when B7-1 (Figure 23A) and B7-2 (Figure 23B) are coated onto plates. Biotinylated mouse CTLA4-Fc fusion protein was incubated with B7-coated plates in the presence of a given concentration of control IgG or the anti-mouse CTLA4 mAbs 9D9 and 9H10. CTLA4 binding was detected using HRP-conjugated streptavidin. Data shown are the average of duplicate determinations and are representative of two independent experiments. Figures 23C and 23D show that 9D9 and 9H10 exhibit differential binding to soluble CTLA4-Fc (Figure 23C) and plate-bound CTLA4-Fc (Figure 23D). Data shown are the average of duplicate determinations and are representative of at least two independent experiments. Figures 23E and 23F show the effect of anti-mouse CTLA4 mAbs 9D9 and 9H10 on the levels of B7-1 (Figure 23E) and B7-2 (Figure 23F) on CD11chi DCs from WT (Ctla4m / m) splenocytes 24 hours after intraperitoneal treatment with 500 μg of antibody. Data are summarized from six independent mice per group in two independent experiments with three mice per group. [Figure 24]Figures 24A and 24B show the effect of 4F10 on the interaction of CTLA4-Fc with plates coated with B7-1 (Figure 24A) or B7-2 (Figure 24B). Biotinylated mouse CTLA4-Fc fusion protein was incubated with B7-coated plates in the presence of a given concentration of control IgG or anti-mouse CTLA4 mAb 4F10. CTLA4 binding was detected using HRP-conjugated streptavidin. Data shown are the average of duplicate determinations and are representative of two independent experiments. Figures 24C and 24D show the effect of 4F10 on B7-1 and B7-2 expression. Summary data for B7-1 (Figure 24C) and B7-2 (Figure 24D) levels from six mice per group. The B7 level in the control IgG-treated group is experimentally defined as 100%. [Figure 25] Adverse effects of chimeric L3D10 and 10D1 in combination with anti-PD-1. The upper panel shows the experimental design. Only 10-week-old female human CTLA4-knockin mice weighing more than 4 grams were used in the study. The indicated proteins or their combinations were administered. Arrows indicate the time of treatment (100 μg / mouse / treatment). Data shown are the mean and standard deviation of % body weight gain. Chimeric L3D10 and 10D1 have comparable cancer therapeutic efficacy in adult mice (Figure 13), but different adverse effects were observed when 10D1 was combined with anti-PD-1 mAb. [Figure 26] Adverse effects of chimeric L3D10 and 10D1 in combination with anti-PD-1. The graph shows the final body weights on day 42 of mice (n=5 / group) from the experiment outlined in Figure 25, which were administered either control IgG, 10D1 + anti-PD-1, or chimeric L3D10 + anti-PD-1. A significant decrease in body weight is observed with the anti-PD1 + 10D1 combination, which was not seen with the anti-PD-1 + chimeric L3D10 combination. [Figure 27]Pathological Effects of Chimeric L3D10 and 10D1 in Combination with Anti-PD-1. To further investigate the relative toxicity of chimeric L3D10 compared to 10D1 when administered in combination with anti-PD-1, we focused on the gross morphology of the mice described in Figure 26 above. The uterus / ovaries / bladder and thymus were significantly smaller in mice treated with 10D1 + PD-1, whereas the organs of mice treated with L3D10 + anti-PD-1 were comparable to those of the hIgG control. In contrast, hearts isolated from mice treated with 10D1 appeared larger in size and had a distinctly white appearance. [Figure 28] Treatment with 10D1 in combination with anti-PD-1 results in abnormal erythropoiesis. Given the cardiac differences observed in Figure 27, we examined erythropoiesis in mice and observed clear differences in mice treated with 10D1 + anti-PD-1 compared with groups treated with L3D10 + anti-PD-1 or control antibody (hIgG), which were very similar. The bone marrow of mice treated with 10D1 + anti-PD-1 was noticeably whitish (Figure 28A), and isolated blood was almost completely white (Figure 28B). Correspondingly, we analyzed erythroid differentiation using the distribution of CD119 and CD71 markers and observed a statistically significant decrease in the number of cells undergoing stage IV development in mice treated with 10D1 + anti-PD-1. Representative FACS profiles are shown in Figure 28C, and summary data are presented in Figure 28D. [Figure 29] Flow cytometry analysis of anti-erythrocyte antibodies. Blood samples from NOD.SCID.Il2rg- / - (NSG) mice were stained with plasma samples from mice treated with antibodies during the perinatal period. Serum from NSG mice and no serum served as negative controls. All sera were used at a 1:50 dilution. These data indicate that none of the mice produced anti-erythrocyte antibodies. [Figure 30]Cardiac pathology in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicity of L3D10 versus 10D1 in combination with anti-PD-1, cardiac histological analysis was performed in the mice described in Figure 26. Mice treated with 10D1 + anti-PD-1 showed high levels of T cell infiltration that was not observed in mice treated with L3D10 + anti-PD-1 or with the human IgG control. [Figure 31] Lung pathology in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicity of L3D10 versus 10D1 in combination with anti-PD-1, lung histology analysis was performed in the mice described in Figure 26. Mice treated with 10D1 + anti-PD-1 showed high levels of T cell infiltration that was not observed in mice treated with L3D10 + anti-PD-1 or with the human IgG control. [Figure 32] Salivary gland pathology in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicity of L3D10 versus 10D1 in combination with anti-PD-1, histological analysis of saliva was performed in the mice described in Figure 26. Mice treated with 10D1 + anti-PD-1 exhibited much higher levels of T cell infiltration than observed in mice treated with L3D10 + anti-PD-1 or the human IgG control. [Figure 33] Kidney and liver pathology in mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. To further determine the toxicity of L3D10 versus 10D1 in combination with anti-PD-1, histological analysis of the kidney and liver was performed in the mice described in Figure 26. Figures 33A-C show kidney sections, and Figures 33D-E show sections taken from the liver. Mice treated with 10D1 + anti-PD-1 exhibited higher levels of T cell infiltration than those observed in mice treated with L3D10 + anti-PD-1 or the human IgG control. [Figure 34]Toxicity scores for mice treated with chimeric L3D10 and 10D1 in combination with anti-PD-1. This histological data, shown in Figures 30-33, is summarized and shows higher toxicity scores for mice treated with 10D1 + anti-PD-1 compared to L3D10 + anti-PD-1, which had slightly higher scores than the hIgG control mouse group. [Figure 35] 10D1 + anti-PD-1 did not have significant toxicity in Ctla4h / m mice, as evidenced by normal weight gain in mice treated with the antibody during the perinatal period. Mice were treated intraperitoneally with a given antibody or combination (100 μg / mouse / injection / antibody) on days 10, 13, 16, 19, and 22. Mice were weighed at least every three days. [Figure 36] L3D10 and 10D1 show similar binding patterns to plate-immobilized CTLA4. ELISA plates were coated with 1 μg / ml of CTLA4-His protein (Sino Biological, China). Biotinylated binding proteins were added at given concentrations, and binding was measured using HRP-conjugated streptavidin. 10D1-1 and -2 are two independent lots of the same antibody. hIgG-Fc is a human Ig negative control. [Figure 37] L3D10 exhibits reduced binding to soluble CTLA4. A given concentration of anti-human CTLA4 mAb was coated onto plates overnight, washed, and blocked with bovine serum albumin. After incubation and washing, 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. [Figure 38]Alignment of the humanized antibody variable regions with the parent L3D10 antibody sequence. The heavy chain variable regions (top) (SEQ ID NOs: 62-64) and light chain variable regions (bottom) (SEQ ID NOs: 70-72) of the humanized antibody sequence are aligned with the parent L3D10 antibody (heavy chain: SEQ ID NO: 57; light chain: SEQ ID NO: 65) and the respective human antibody frameworks (heavy chain: SEQ ID NOs: 58-61; light chain: SEQ ID NOs: 66-69). Backmutations relative to the murine parent sequence are highlighted in yellow. Novel amino acids, i.e., amino acid residues not present in the parent antibody sequence or the respective human antibody frameworks, are highlighted in green. Mutations introduced into the CDR2 sequence are shown in purple. The CDR sequences are shown in red based on 1528427351721_0. [Figure 39] Antitumor activity of humanized L3D10 antibody compared to 10D1. Using the MC38 mouse tumor model with human CTLA4 knock-in mice, the antitumor activity of humanized L3D10 antibody was examined compared to chimeric L3D10 antibody and 10D1. The upper panel shows the treatment schedule for the in vivo experiment: mice were administered a total of four doses of antibody every three days starting on day 7 after inoculation. All humanized antibodies (n=6 / group) completely eradicated tumors, comparable to 10D1 (lower panel). [Figure 40] Antitumor activity of humanized L3D10 antibody in CTLA4h / m mice. The upper panel shows the treatment schedule for the in vivo experiment: CTLA4h / m mice were administered 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 the indicated days after MC38 tumor injection. Tumor size was measured every 3 days. [Figure 41]Therapeutic efficacy of anti-CTLA-4 mAb in the minimally invasive B16-F1 tumor model. The antitumor activity of the humanized L3D10 antibody was investigated using the B16-F1 mouse tumor model, a mouse model of human CTLA4 knock-in mice. 1 × 10 B16 tumor cells were injected subcutaneously into Ctla4 h / h mice (n = 5–6). On days 2, 5, and 8, mice were treated with control Ig, 10D1, chimeric L3D10, or PP4637 and PP4638 (250 μg / mouse, intraperitoneally). 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 the mean ± SEM of 5-6 mice per group. Tumor size was calculated as 0 for mice that did not develop any tumors. [Figure 42] Comparison of 10D1, PP4631, and PP4637 females for toxicity in combination with anti-PD-1 mAb. Female CTLA4h / h mice were treated with four injections of antibody (100 μg / mouse / injection, once every three days) or control Fc on postnatal day 10 or 11, as indicated in the legend. Mice were weighed every three days. Data shown are the mean and SEM of % weight gain over a 30-day period. All mice were sacrificed on day 43 for histological analysis. The number of mice used per group is indicated in parentheses in the labels. [Figure 43] Combination therapy with 10D1 and anti-PD-1 induces anemia, whereas treatment with either PP4631 + anti-PD-1 or PP4637 + anti-PD-1 does not. Data shown are hematocrits from 43-day-old mice that received four antibody treatments at a dose of 100 μg / mouse / antibody on days 11, 14, 17, and 20. [Figure 44A]Combination therapy with 10D1 plus anti-PD-1 induces systemic T cell activation, whereas combination therapy with either PP4631 plus anti-PD-1 or PP4637 plus anti-PD-1 does not. Data shown are the percentages of CD4 (upper panel) and CD8 T cells (lower panel) with naive (CD44loCD62Lhi), central memory (CD44hiCD62Lhi), and effector memory (CD44hiCD62Llo) phenotypes in either peripheral blood (Figure 44A) or spleen (Figure 44B). Cells were harvested from 43-day-old mice that received four antibody treatments at a dose of 100 μg / mouse / antibody on days 11, 14, 17, and 20. [Figure 44B] Combination therapy with 10D1 + anti-PD-1 results in systemic T cell activation, whereas combination therapy with either PP4631 + anti-PD-1 or PP4637 + anti-PD-1 does not. Data shown are the percentage of CD4 (upper panel) and CD8 T cells (lower panel) with naive T cell (CD44loCD62Lhi), central memory T cell (CD44hiCD62Lhi), and effector memory T cell (CD44hiCD62Llo) phenotypes in either peripheral blood (Figure 44A) or spleen (Figure 44B). Cells were harvested from 43-day-old mice that received four antibody treatments at a dose of 100 μg / mouse / antibody on days 11, 14, 17, and 20. [Figure 45] Humanization of L3D10 does not affect binding to immobilized CTLA4. The ability of the humanized L3D10 antibodies to bind to immobilized CTLA4 was determined as described in Figure 36. The X-axis indicates the concentration of anti-CTLA-4 mAb added to the solution. Humanization did not affect binding to immobilized CTLA4, with all three humanized antibodies showing similar binding to the parent chimeric L3D10 antibody and 10D1. A similar pattern was observed when CTLA4-Ig was used instead of CTLA-4-his. [Figure 46]Humanization further reduces binding of L3D10 to soluble CTLA4. The ability of humanized L3D10 antibodies to bind to soluble CTLA4 was determined as described in Figure 37. The X-axis represents the concentration of anti-CTLA-4 mAb coated onto the ELISA plate. Humanization further reduces binding to soluble CTLA4 compared to the parent L3D10 chimeric antibody. A similar pattern was observed when CTLA4-Ig was used instead of CTLA-4-his. [Figure 47] PP4631, PP4638, and PP4637 do not block B7-CTLA-4 interaction in vitro. Figure 47A shows blockade of B7-1-CTLA-4 interaction by anti-human CTLA-4 mAbs 10D1, PP4631, PP4637, and L3D10. B7-1Fc was immobilized at a concentration of 0.5 μg / ml. Biotinylated CTLA4-Fc was added at 0.25 μg / ml along with the given dose of antibody. Data shown are the mean optical density values ​​measured in duplicate at 405 nM. Figure 47B shows blockade of B7-2-CTLA-4 interaction by anti-human CTLA-4 mAbs 10D1 and L3D10. Similar to Figure 47A, except that B7-2-Fc was immobilized. [Figure 48] PP4631 and PP4637 do not block B7-CTLA-4 interactions in vivo, as evidenced by their lack of effect on B7-1 and B7-2 expression on dendritic cells. Summary data for B7-1 (a) and B7-2 (b) levels from three mice per group. B7 levels in the control IgG-treated group were experimentally defined as 100%. [Figure 49] PP4637, which exhibits the best safety profile when combined with anti-PD-1 mAbs (see Figure 42), is the most potent at inducing tumor rejection based on tumor rejection at the lowest therapeutic dose. CTLA4 h / m mice were administered control IgFc or one of three different anti-human CTLA4 mAbs at doses of 30 (-30, solid line) or 10 (-10, dotted line) μg / injection on the days indicated. Tumor size was measured every three days. At 10 μg / injection, PP4637 (HL32) is the most efficient at inducing tumor rejection. [Figure 50] Assessment of humanized antibody purity. Transiently expressed humanized L3D10 antibodies were purified by Protein A chromatography, and samples from all three antibodies were analyzed by reducing and non-reducing SDS-PAGE. The purified proteins produced gel bands indicative of the size of the antibody molecule under both reducing and non-reducing conditions. The "flow-through" lane represents the Protein A column flow-through, suggesting that the majority of the antibody Protein A was attached to the Protein A column. [Figure 51] Size-exclusion chromatography (SE-HPLC) of transiently expressed proteins. Protein samples of each humanized antibody were analyzed by SE-HPLC followed by one-step Protein A chromatography. Top panel: antibody PP4631. Middle panel: antibody PP4637. Bottom panel: antibody PP4638. [Figure 52] CE-SDS analysis of transiently expressed proteins. Protein samples of each humanized antibody were analyzed by CE-SDS followed by one-step Protein A chromatography. The left panel shows the results under non-reducing conditions, and the right panel shows the results under reducing conditions. Top panel: antibody PP4631. Middle panel: antibody PP4637. Bottom panel: antibody PP4638. [Figure 53A] Charge isoform profile and deamidation of humanized L3D10 antibody determined by capillary isoelectric focusing (cIEF). The deamidation level of the protein under high pH stress was determined by comparing the humanized L3D10 antibody before and after high pH stress treatment for two different periods (5 hours and 12.5 hours) and analyzed by cIEF analysis. Figures 53A-53C show the profiles of antibodies PP4631, PP4637, and PP4638, respectively. [Figure 53B]Charge isoform profile and deamidation of humanized L3D10 antibody determined by capillary isoelectric focusing (cIEF). The deamidation level of the protein under high pH stress was determined by comparing the humanized L3D10 antibody before and after high pH stress treatment for two different periods (5 hours and 12.5 hours) and analyzed by cIEF analysis. Figures 53A-53C show the profiles of antibodies PP4631, PP4637, and PP4638, respectively. [Figure 53C] Charge isoform profile and deamidation of humanized L3D10 antibody determined by capillary isoelectric focusing (cIEF). The deamidation level of the protein under high pH stress was determined by comparing the humanized L3D10 antibody before and after high pH stress treatment for two different periods (5 hours and 12.5 hours) and analyzed by cIEF analysis. Figures 53A-53C show the profiles of antibodies PP4631, PP4637, and PP4638, respectively. [Figure 54] Differential scanning calorimetry (DSC) thermal analysis of humanized L3D10 antibody. To determine the thermal stability and melting temperatures of different antibodies, they were subjected to differential scanning calorimetry (DSC) thermal analysis. Figures 54A-54C show the normalized DSC curves of antibodies PP4631, PP4637, and PP4638, respectively. [Figure 55]Alignment of the human, macaque, and mouse CTLA-4 extracellular domains. 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 are aligned, with conserved amino acids (relative to the human sequence) indicated by dashed lines (-). To aid in alignment, the mouse sequence has deletions and insertions (relative to the human and monkey sequences) at positions highlighted in yellow. The known B7-1Ig binding site is shown in bold and underlined. The sequences show that the human and monkey sequences are highly conserved, while the mouse sequence has numerous amino acid differences. Based on this sequence alignment, 11 mutant (M1-M11) (SEQ ID NOs: 40-50) human CTLA-4 Fc proteins incorporating mouse-specific amino acids were designed; the amino acids incorporated into each mutant protein are indicated in blue. [Figure 56A] Amino acid sequence composition of WT and mutant CTLA-4Fc proteins. DNA constructs encoding the WT CTLA-4Fc protein (SEQ ID NO: 39) and 11 mutant proteins (SEQ ID NOs: 40-50) incorporating mouse Ctla-4 amino acids were designed as shown. The amino acid sequence is of the mature protein including the IgG1 Fc portion, not the signal peptide. The known B7-1Ig binding site is shown in large blue letters and double underlined. The mouse amino acid residues substituted in the mutants are shown in red subscript. The IgG1 Fc portion of the protein is underlined. [Figure 56B] Amino acid sequence composition of WT and mutant CTLA-4Fc proteins. DNA constructs encoding the WT CTLA-4Fc protein (SEQ ID NO: 39) and 11 mutant proteins (SEQ ID NOs: 40-50) incorporating mouse Ctla-4 amino acids were designed as shown. The amino acid sequence is of the mature protein including the IgG1 Fc portion, not the signal peptide. The known B7-1Ig binding site is shown in large blue letters and double underlined. The mouse amino acid residues substituted in the mutants are shown in red subscript. The IgG1 Fc portion of the protein is underlined. [Figure 57]Mutations in M11 (AA103-106, YLGI>fcGm) selectively abolish antibody binding to human CTLA-4. Data shown are the average of duplicate determinations and show binding of B7-1Fc (a), L3D10 (b), PP4631 (c), and PP4637 (d) to plate-coated hCTLA4-Fc (open circles), mCTLA4-Fc (closed triangles), M11 (closed circles), and IgG1-Fc (open triangles). [Figure 58] In the 3D structure of the B7-1-CTLA4 complex, L3D10, PP4631, and PP4637 are mapped to epitopes adjacent to the B7-1 binding site. The B7-1 binding motif is colored red, and the antibody epitope is colored purple. B7-1 is depicted as a space-filled ribbon above CTLA4, while CTLA-4 is depicted as an unfilled ribbon. [Figure 59] Amino acid sequence composition of WT (SEQ ID NO: 39) and mutant CTLA-4 Fc proteins M12-M17 (SEQ ID NOs: 51-56). DNA constructs encoding six mutant CTLA-4 Fc proteins M12-M17 incorporating mouse Ctla-4 amino acids were designed as shown. The amino acid sequence is of the mature protein including the IgG1 Fc portion, not the signal peptide. The known B7-1Ig binding site is shown in large blue letters and double underlined. The mouse amino acid residues substituted in the mutants are shown in red subscript. The IgG1 Fc portion of the protein is underlined. [Figure 60] Mutational analysis reveals distinct binding requirements for 10D1 (Figure 60A), PP4631 (Figure 60B), and PP4637 (Figure 60C) for CTLA-4. CTLA-4 Fc variants were coated at 1 μg / ml overnight at 4°C. After blocking with BSA, a given concentration of biotinylated anti-CTLA-4 mAb was added and incubated for 2 hours. After washing away unbound antibody, bound antibody was detected with HRP-conjugated streptavidin. [Figure 61]Therapeutic effects of anti-4-1BB and anti-CTLA-4 antibodies in both minimal disease (Figure 61A) and established tumor (Figure 61B) models. Figure 61A shows treatment of minimal disease. C57BL / 6 mice were subcutaneously inoculated with 5 × 10 MC38 cells. Control hamster and rat IgG, anti-CTLA-4, and / or anti-4-1BB antibodies were injected on days 2, 9, and 16 after tumor cell injection. Tumor size was measured by physical examination. Data shown are tumor growth kinetics; each line represents tumor growth in one mouse. The presented size is the product of the longest and shortest diameters of the tumor. Figure 61B shows treatment of established tumors. This is similar to Figure 61A, except that treatment began 14 days after tumor challenge. All mice had established tumors ranging in size from 9 to 60 mm before treatment with the mAbs began. The combined effect of the two antibodies on established tumors was repeated three times. [Figure 62] CD8 T cells, but not CD4 or NK cells, are essential for antibody-induced tumor rejection. Tumor-bearing mice were depleted of CD4, CD8, or NK cells with three injections of antibodies specific for either CD4, CD8, or NK1.1 on days 9, 12, and 16 after tumor cell inoculation (*). Therapeutic antibodies (anti-CTLA-4 + anti-4-1BB) were injected on days 9, 16, and 23 (vertical arrows). Data shown are the mean and SEM of tumor size (n = 3). P < 0.05 for the CD8-depleted group compared with each of the other groups (†). [Figure 63]Combination therapy reduced the host response to anti-CTLA-4 antibodies. Hamster anti-mouse CTLA-4 (Figure 63A) or rat anti-mouse 4-1BB (Figure 63B) antibodies were coated onto ELISA plates. Different dilutions of serum from each group of five mice were added to the plates. The relative amount of bound antibody was determined using a second-stage reagent (biotinylated goat anti-mouse antibody depleted of reactivity to rat and hamster IgG by absorbance). Data shown are the mean and SEM of optical densities at 490 nm. Similar reductions in host antibody responses to anti-CTLA-4 and 4-1BB were observed when tumor-free mice were treated with the same antibodies (data not shown). [Figure 64] Combination therapy using anti-4-1BB and L3D10 (anti-human CTLA4) antibodies in human CTLA-4 gene knockin mice. Figure 64A shows the therapeutic effect. Human CTLA4 knockin mice were subcutaneously inoculated with 5 × 10 MC38 tumor cells. 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 groups treated with the two antibodies showed a significant reduction in tumor size compared to the control (P < 0.0001), or treatment with L3D10 antibody (P = 0.0007), or anti-4-1BB antibody (P = 0.03). When the control IgG-treated group reached the initial clearance criterion, all tumor-bearing mice were sacrificed. Figure 64B shows persistent immunity in mice receiving combination therapy. Tumor-free mice in the two-antibody treated group developed persistent immunity against MC38 tumors. 110 days after the first tumor cell challenge, tumor-free mice treated with the two antibodies or control naive mice were subcutaneously injected with 5 x 10 tumor cells. Tumor growth was monitored by physical examination. All mice that initially rejected tumors were completely resistant to rechallenge, whereas all naive mice showed progressive tumor growth. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition As used herein, the term "antibody" is intended to mean an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" is intended to distinguish that domain of an immunoglobulin from the domain broadly shared by antibodies (the antibody Fc domain). The variable region contains a "hypervariable region" whose residues are involved in antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., typically about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and about residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Reference 44) and / or residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Reference 45). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein. The term antibody includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to an antibody of the invention). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0029] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody containing the antibody's complementarity-determining regions ("CDRs") and, optionally, framework residues comprising the antibody's "variable region" antigen recognition site, which exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab').sub.2, Fv, single chain (ScFv), and mutants, naturally occurring variants thereof, as well as fusion proteins comprising the antibody's "variable region" antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor, or a receptor ligand, etc.). As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0030] Human, chimeric, or humanized antibodies are particularly preferred for in vivo use in humans, although murine antibodies or antibodies from other species may be advantageously utilized for many applications (e.g., in vitro or in situ detection assays, acute in vivo uses, etc.).

[0031] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Chimeric antibodies comprising one or more CDRs from a non-human species and a framework region from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including CDR grafting (European Patent No. EP 239,400, International Publication No. WO 91 / 09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent No. EP 592,106, European Patent No. EP 519,596; 46-48), and chain shuffling (U.S. Patent No. 5,565,332).

[0032] The present invention particularly relates to "humanized antibodies." As used herein, the term "humanized antibody" refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Thus, all portions of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding portions of a native human immunoglobulin sequence. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody does not encompass a typical chimeric antibody, since the entire variable region of a chimeric antibody is non-human. A donor antibody is said to be "humanized" by the process of "humanization" because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody providing the CDRs. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.A humanized antibody optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that has been altered by substitution, deletion, or introduction of additional amino acid residues (i.e., mutation) that immunospecifically binds to an FcγRIIB polypeptide.

[0033] Detailed Description Antibodies against the human CTLA4 protein ipilimumab have been shown to improve the survival of cancer patients as sole immunotherapeutic agents, in combination with other therapeutic agents, such as, but not limited to, anti-PD-1 antibodies (13-15). However, the therapeutic effect is 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 surprisingly discovered that anti-CTLA4 antibodies can be used to induce cancer rejection while also reducing the autoimmune adverse effects associated with immunotherapy.

[0034] Antibody compositions and antigen-binding fragments thereof are provided herein. The invention further relates to embodiments of such molecules, wherein the molecules are monoclonal antibodies, human antibodies, chimeric antibodies, or humanized antibodies.

[0035] Specifically, the present invention provides molecules comprising an antigen-binding fragment of an antibody that immunospecifically binds to CTLA4, particularly human CTLA4, preferably expressed on the surface of a living cell at endogenous or transfected levels. The present invention particularly relates to embodiments of such molecules in which the antigen-binding fragment binds to CTLA4 and the living cell is a T cell.

[0036] The present invention relates to antibodies and antigen-binding fragments thereof capable of immunospecifically binding to CTLA4. In some embodiments, such molecules are capable of blocking the binding of B7.1 and B7.2 to CTLA4.

[0037] The present invention further relates to embodiments of such molecules in which the molecules are monoclonal, human, chimeric, or humanized antibodies. The present invention includes embodiments in which such antibodies are monospecific, bispecific, trispecific, or multispecific.

[0038] The invention further relates to embodiments of molecules or antibodies that bind to CTLA4, the antigen-binding fragment of which comprises six CDRs, the CDRs comprising the CDRs of the anti-CTLA4 antibody L3D10. Specifically, the antibody comprises the three light chain CDRs and the three heavy chain CDRs of the anti-CTLA4 antibody L3D10.

[0039] The present invention further relates to embodiments of the aforementioned antibodies, wherein the antibody is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, or receptor ligand.

[0040] The present invention further relates to pharmaceutical compositions comprising a therapeutically effective amount of any of the aforementioned antibody compositions and a physiologically acceptable carrier or excipient. Preferably, the compositions of the present invention comprise a prophylactically or therapeutically effective amount of a humanized antibody of the present invention and a pharmaceutically acceptable carrier.

[0041] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0042] Generally, the components of the composition of the present invention can be supplied separately or mixed together in unit dosage form, for example, as dry lyophilized powder or water-free concentrate in a sealed container such as an ampoule or a small sachet indicating the amount of active ingredient.When the composition is administered by infusion, it is dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0043] The compositions of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0044] The present invention further relates to the use of the antibody compositions and pharmaceutical compositions thereof described herein for upregulating immune responses. Upregulation of the immune system is particularly desirable in the treatment of cancer and chronic infectious diseases, and the present invention therefore has utility in the treatment of such diseases. As used herein, the term "cancer" refers to a neoplasm or tumor that results from the abnormal, uncontrolled proliferation of cells. As used herein, cancer explicitly includes leukemia and lymphoma. This term refers to diseases involving cells that have the potential to metastasize to distant sites.

[0045] Accordingly, the methods and compositions of the present invention may also be useful in the treatment or prevention of a wide variety of cancers or other abnormal proliferative disorders, including (but not limited to): carcinomas, including those of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic malignancies of the lymphatic system, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; acute and chronic myeloid leukemia, etc. Hematopoietic tumors of the myeloid lineage, including promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma. Cancers caused by abnormalities in apoptosis are also contemplated for treatment by the methods and compositions of the present invention. Such cancers may include, but are not limited to, follicular lymphoma, carcinoma due to p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndromes. In certain embodiments, malignant tumors or neoplastic changes (such as metaplasia and dysplasia), or hyperproliferative disorders are treated or prevented by the methods and compositions of the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by the methods and compositions of the invention.

[0046] In another embodiment of the present invention, the antibody compositions and antigen-binding fragments thereof can be used in conjunction with other anti-tumor therapies, including, but not limited to, current standard and experimental chemotherapy, hormonal therapy, biological therapy, immunotherapy, radiation therapy, or surgery. In some embodiments, the molecules of the present invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more drugs, therapeutic antibodies, or other agents known to those skilled in the art for the treatment and / or prevention of cancer, autoimmune disease, infectious disease, or poisoning. Such drugs include, for example, any of the biological response modifiers, cytotoxins, antimetabolites, alkylating agents, antibiotics, or antimitotic agents, and immunotherapies discussed above.

[0047] In a preferred embodiment of the present invention, the antibody compositions and antigen-binding fragments thereof can be used in combination with other anti-tumor immunotherapeutics. In such embodiments, the molecules of the present invention are administered in combination with molecules that interfere with or enhance alternative immunoregulatory 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 that modulate the activity of effector molecules such as cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-β, IFNg, Flt3, BLys) and chemokines (e.g., CCL21) to enhance immunomodulatory effects. Certain embodiments include bispecific antibodies comprising the anti-CTLA4 antibody compositions described herein and anti-PD-1 (pembrolizumab (Keytruda) or nivolumab (Opdivo)), anti-B7-H1 (atezolizumab (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 yet another embodiment, the molecules of the invention are administered in combination with molecules that activate different stages or aspects of the immune response to achieve a broader immune response. In a more preferred embodiment, the antibody compositions and antigen-binding fragments thereof are used in combination with anti-PD-1 or anti-4-1BB antibodies without exacerbating autoimmune side effects.

[0048] Another embodiment of the present invention includes bispecific antibodies comprising an antibody that binds CTLA4 cross-linked to an antibody that binds another immune stimulator. Particular 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 present invention further relates to the use of such antibodies to treat cancer.

[0049] Methods of administering antibody compositions of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In specific embodiments, antibodies of the invention are administered intramuscularly, intravenously, or subcutaneously. Compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), and may be administered together with other biologically active substances. Administration may be systemic or local.

[0050] Yet another embodiment of the present invention relates to monitoring the in vivo blocking effect of anti-CTLA4 antibodies 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 and suppresses autoimmune diseases by downregulating the expression of B7-1 and B7-2 on APCs, such as dendritic cells. Thus, upregulation of the B7 molecules B7.1 and B7.2 can be an indication of in vivo blockade of the B7-CTLA4 interaction. In certain embodiments, peripheral or intratumoral immune cells are removed from a subject before and after anti-CTLA4 treatment and assayed ex vivo for a reduction in B7.1 and / or B7.2 levels on the surface of the immune cells. The presence of the blocking anti-CTLA4 antibody prevents the binding of B7.1 / B7.2 by 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 are measured on antigen-presenting cells. In a most preferred embodiment, the levels of B7.1 and B7.1 are measured on dendritic cells.

[0051] In a further embodiment, changes (decreases) in B7.1 and B7.2 on immune cells after anti-CTLA4 treatment are used as biomarkers to measure the in vivo biological activity of anti-CTLA4 antibodies, to monitor the positive response to anti-CTLA4 treatment by measuring the expression levels of B7.1 and / or B7.2 on immune cells, and to compare expression levels before and after treatment. In a preferred embodiment, the expression levels of B7.1 and / or B7.2 are monitored over time during the course of anti-CTLA4 treatment. [Example]

[0052] Example 1. Production of chimeric anti-CTLA4 antibodies Using human CTLA4 gene knock-in mice and hu-PBL-Scid mice, murine anti-human CTLA4 antibodies have previously been shown to suppress tumor growth, and L3D10 was identified as the most effective mAb tested. However, none of the resulting antibodies were able to achieve complete tumor rejection, even when administered at relatively high doses (>10 mg / kg) before the formation of palpable tumors (as early as day 2) after tumor cell challenge (19-21).

[0053] Because the murine antibody is an IgG1 subclass antibody that lacks strong antibody-dependent cellular cytotoxicity (ADCC), and because ADCC may be involved in tumor rejection, the Fc of the mAb was modified in several ways to achieve better immunotherapeutic efficacy. First, the murine IgG1, which has poor ADCC activity, was replaced with human IgG1, which has strong ADCC activity, to generate a chimeric antibody. Second, based on previously published techniques (22), three mutations (S298A, E333A, and K334A) were introduced into the CH to increase ADCC activity. Third, three mutations (M252Y, S254T, and T256E) were introduced to extend the half-life of the antibody in vivo (23). The design of the new chimeric antibody is shown in the left panel of Figure 1.

[0054] To engineer the antibodies, the variable regions of the L3D10 hybridoma were first identified by DNA sequencing using standard methods known in the art. The nucleotide sequences were translated into amino acids as set forth in SEQ ID NO:1 and SEQ ID NO:2. The normal human IgG1 Fc sequence and the mutant Fc sequence are disclosed in SEQ ID NO:3 and SEQ ID NO:4, respectively. The amino acid and codon-optimized nucleotide sequences of the heavy and light chain sequences are disclosed in SEQ ID NO:5-8.

[0055] DNA corresponding to SEQ ID NO:5 and SEQ ID NO:7 was synthesized and inserted into an expression vector, and the vector was transfected into HEK293 cells using the designed sequence. Briefly, HEK293 cells were seeded into shake flasks one day before transfection and grown in serum-free, chemically defined medium. Using standard operating procedures for transient transfection, DNA expression constructs were transiently transfected into 0.5 liters of suspension HEK293 cells. After 20 hours, cell samples were taken for viability and viable cell count, and titers were measured (Octet QKe, ForteBio). Further measurements were performed throughout the transient transfection production run. Culture medium was harvested on day 5. Conditioned medium for L3D10 was collected and clarified from the transient transfection production run by centrifugation and filtration. The supernatant was loaded onto a Protein A column and eluted with a low pH buffer. Filtration was performed using a 0.2 μm membrane filter before aliquoting. After purification and filtration, the protein concentration was calculated from OD280 and the extinction coefficient. A total of 43.2 mg of Ig protein was obtained from one transfection.

[0056] Example 2. The binding site of the chimeric L3D10 antibody only partially overlaps with 10D1 In the clinic, the anti-CTLA4 antibody ipilimumab has been shown to improve survival in cancer patients but induce significant autoimmune adverse effects. To assess the equivalent binding sites of the chimeric L3D10 antibody and 10D1, we compared their binding to CTLA4 and their ability to compete for binding to CTLA4. While both antibodies bind to immobilized CTLA4 protein with comparable efficiency (Figure 2), 10D1 does not completely block the binding of chimeric L3D10 to CTLA4 (Figure 3). As expected, unlabeled L3D10 completely blocks the binding of labeled L3D10, suggesting that the antibody binding sites of L3D10 and 10D1 only partially overlap.

[0057] Example 3. CTLA4:B7.1 and CTLA4:B7.2 interactions are more efficiently blocked by chimeric L3D10 antibody rather than 10D1 It has been reported that the anti-human CTLA4 mAb 10D1 can block B7-CTLA4 interactions when soluble B7-1 and B7-2 are used to interact with immobilized CTLA4 (49). Because B7-1 and B7-2 function as cell surface costimulatory molecules, we used immobilized B7-1 and B7-2 to assess the ability of anti-CTLA4 antibodies to block B7-CTLA4 interactions. Using a competitive ELISA assay format, we assessed the ability of L3D10 and 10D1 to block the binding of the CTLA4 fusion protein CTLA4-Ig to both plate-immobilized B7.1 and B7.2 and cell membrane-expressed B7.1 and B7.2. For these experiments, we used a chimeric anti-human CTLA4 mAb with affinity (2.3 nM) similar to that of 10D1 (4 nM) (49). For the plate-immobilized assay, we used B7.1Fc or B7.2Fc at 400 uM. oBiotinylated CTLA4-Fc was coated onto ELISA plates at 1 μg / ml for overnight at 4°C or for 2 hours at 37°C. Biotinylated CTLA4-Fc was mixed with a given concentration of either B7.1-Fc, 10D1, or chimeric L3D10. Horseradish peroxidase-conjugated streptavidin was used to determine the amount of CTLA4-Fc bound to B7.1 on the plate. As shown in Figure 4, chimeric L3D10, B7.1Fc, and CTLA4-Fc all efficiently blocked the CTLA4-Fc:B7.1 interaction, whereas two separate material lots of 10D1 failed to block the interaction. L3D10 showed significant blocking of plate-immobilized B7.1 binding at concentrations as low as 0.2 μg / ml, with 50% inhibition (IC ) at around 3 μg / ml. 50 Similarly, L3D10 achieves an IC of 0.03 μg / ml. 50 10D1 from two different material lots blocked CTLA4-Fc binding to plate-immobilized B7.2 at IC values ​​of approximately 200 μg / ml. 50 showed minimal blockage (Fig. 5). However, consistent with a previous report (49), antibody 10D1 potently inhibited the B7-1-CTLA4 interaction in the reverse experiment when plate-immobilized CTLA4 was used to interact with soluble B7-1 (Fig. 6).

[0058] In cell membrane protein binding experiments, when B7.1 was expressed on the surface of CHO cells, L3D10 blocked CTLA4-Fc binding, whereas 10D1 from two different material lots did not, even when used at 512 μg / ml (Figure 7). 10D1, which is much weaker than L3D10 but at higher doses, achieved approximately 25% blockade between human CTLA4 and murine B7-1 (Figure 8). In the case of B7.2 expressed on the surface of CHO cells, L3D10 also blocked, whereas 10D1 only partially blocked; less than 50% inhibition was observed, even when 10D1 was used at 512 μg / ml (Figure 9).

[0059] A potential caveat is that biotinylation may have affected the binding of 10D1 to CTLA4-Fc. To address this issue, we compared the binding of L3D10 and 10D1 to biotinylated CTLA4-Fc, which was used in blocking studies. As shown in Figure 10, 10D1 is more effective than L3D10 at inhibiting biotinylated CTLA4-Fc. Therefore, the failure of 10D1 to block was not due to insufficient binding to biotinylated CTLA4-Fc. A similar pattern is observed when polyhistidine-tagged CTLA4 is used to interact with CHO cells transfected with human B7-1 (Figure 11). Taken together, our data suggest that the ability of antibody 10D1 to block B7-CTLA4 interaction is highly dependent on the assay used, with immobilized B7-1 and B7-2 showing minimal to undetectable blocking activity, and that antibody L3D10 is a potent blocker of B7-CTLA4 interaction regardless of whether the B7 proteins are immobilized.

[0060] Example 4. Chimeric L3D10 antibodies induce tumor rejection more efficiently than unmodified L3D10 It was previously reported that murine L3D10 failed to induce complete remission of MC38 tumors, although a significant delay was observed (19, 20). To determine whether chimeric L3D10 could induce complete remission in syngeneic mice, 1 × 10 6 MC38 tumor cells were implanted into syngeneic C57BL / 6 mice. One week later, when tumors reached approximately 5 mm in diameter, the mice were treated with either control IgG or chimeric L3D10 mAb at doses only half the dose used in previous studies with murine L3D10. As shown in Figure 12, despite the potential immunogenicity of the human Ig sequence, we found that chimeric L3D10 induced complete remissions in all mice tested. Because treatment was initiated after a large tumor burden was established, which is much more difficult than when tumors were not palpable (19), these experiments demonstrate that chimeric L3D10 is more efficient than unmodified L3D10.

[0061] Example 5. Chimeric L3D10 antibody is equally active as 10D1 in inducing tumor rejection The availability of human CTLA4 gene knock-in mice (20) provided an unprecedented opportunity to test the biological activity of chimeric anti-human CTLA-4 antibodies using 10D1, a clinically used anti-CTLA-4 mAb. In this humanized mouse model, the CTLA4 gene, encoding a product with 100% identity to the human CTLA-4 protein, was 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 of human CTLA4-knockin mice, it was clear that both antibodies were equivalent in inducing tumor rejection, although tumors grew progressively in the IgG control group. Figure 13 shows the effect of antibody treatment on tumor size from two experiments.

[0062] An intriguing question is whether anti-CTLA-4 mAbs need to interact with all CTLA-4 (i.e., achieve target saturation) to exert their immunotherapeutic effects. h / h F1 mice from the CTLA4 m / m Both mice express mouse and human CTLA-4 proteins in a codominant manner. Interestingly, as shown in Figure 14, both chimeras, L3D10 and 10D1, effectively induced tumor rejection, even though approximately 50% of the CTLA-4 protein (i.e., the mouse version of the protein) cannot be bound by anti-human CTLA-4 mAb. Importantly, in this setting, i.e., when gene dose is limited, L3D10 is more therapeutically effective than 10D1 (P<0.05).

[0063] Previous studies have shown that anti-mouse CTL1A-4 mAb cannot induce rejection of the melanoma cell line B16-F1 unless combined with other therapeutic modalities. Therefore, we also tested the antitumor effects of chimeric L3D10 and 10D1 antibodies using the more challenging B16 tumor model in human CTLA4 knock-in mice. As shown in Figure 15, neither L3D10 nor ipilimumab can induce rejection of established tumors, and both induce statistically significant tumor growth delays, although the difference between the different antibodies is not statistically significant.

[0064] Example 6: Blockade of CTLA4 in vivo CTLA4 is primarily expressed among Tregs and suppresses autoimmune diseases by downregulating B7-1 and B7-2 expression on dendritic cells (50). Because targeted mutation of Ctla4 (50) and treatment with a blocking anti-CTLA4 mAb (51) upregulated B7-1 and B7-2 expression on dendritic cells, it has been proposed that the physiological function of CTLA4 on Tregs is to downregulate B7 on DCs. Therefore, using the upregulation of B7 as a measure of in vivo blockade of the B7-CTLA4 interaction, we investigated the function of Ctla4 with a homozygous knock-in of the human CTLA4 gene. h / h An assay was developed using T cells from mice.

[0065] As outlined in Figure 16, surface-expressed B7.1 or B7.2 binds to CTLA4 on the surface of T cells, resulting in downregulation of B7.1 and B7.2 expression. However, binding of a blocking anti-CTLA4 antibody prevents B7.1 / B7.2 binding, which prevents downregulation of B7.1 and B7.2, resulting in a net increase in B7.1 / B7.2 expression. However, when chimeric T cells expressing both human and mouse CTLA4 are used, antibodies that bind to human CTLA4 do not prevent B7.1 / B7.2 binding to mouse CTLA4, and the inhibition of B7.1 / B7.2 is restored.

[0066] CTLA4-humanized mice have been described, which express a CTLA4 gene with 100% identity to the human CTLA4 protein under the control of the endogenous mouse Ctla4 locus (20). Homozygous knock-in mice (CTLA4 h / h ) were backcrossed onto the C57BL / 6 background for at least 10 generations. h / h Heterozygous mice (CTLA4) were generated by crossing WT BALB / c mice with CTLA4 mice. h / m ) was produced.

[0067] To test the clinically proven therapeutic agent, anti-CTLA4 mAb 10D1, a very high dose of anti-CTLA4 mAb (500 μg / mouse, which is approximately 25 mg / kg, or 8-fold the highest dose used in the clinic) was administered to CTLA4 h / h or Ctla4 m / h Mice were injected, and spleen cells were harvested 24 hours after injection to detect Cd11c hi The levels of B7-1 and B7-2 on DCs were measured (Figures 17A-B). As shown in Figures 17C-E, Ctla4 cells administered human IgG1-Fc showed a significant increase in the levels of B7-1 and B7-2 on DCs. h / h Compared to mice treated with chimeric L3D10, DCs from mice treated with chimeric L3D10 showed a statistically significant increase in B7.1 expression in T cells expressing human CTLA4, but not in T cells expressing both human and mouse CTLA4. Similar results were observed for B7.2, as shown in Figures 17C–E. The degree of B7-2 upregulation is comparable to that obtained using a blocking anti-CTLA4 mAb in human Treg-DC cocultures (66).

[0068] To further confirm the specificity of the in vivo assay, we used Ctla4, a gene that codominantly expresses mouse and human CTLA4. m / h We investigated whether L3D10 could upregulate B7 in mice. Because at least 50% of CTLA4 does not bind to anti-human CTLA4 antibodies, we expected that they would not be very potent in blocking the B7-CTLA4 interaction. Indeed, neither antibody upregulated CTLA4.m / h Ctla4 did not result in upregulation of B7-1 and B7-2 on DCs from mice (Fig. 17C, D, F). m / h The complete absence of blockade by L3D10 in mice suggests that CTLA4 encoded by a mouse allele that does not bind L3D10 (Figure 18) is sufficient to downregulate B7 expression. Thus, our data show that 10D1, at doses at least 8-fold higher than the highest dose used in the clinic, does not block the B7-CTLA4 interaction when B7 is plate-immobilized or anchored to a cell membrane, both in vivo and in vitro.

[0069] Ctla4 m / h The complete absence of blockade by L3D10 in mice suggests that CTLA4 encoded by a mouse allele that does not bind L3D10 (Figure 18) is sufficient to downregulate B7 expression. In contrast, 10D1 did not increase B7.1 or B7.2 expression. Based on the model, this suggests that L3D10, but not 10D1, blocks CTLA4 activity in vivo. However, despite their apparent differences in blocking activity, both L3D10 and 10D1 inhibited the chimeric CTLA4 m / h They exhibited potent antitumor activity in the MC38 mouse model. While tumors grew progressively in mice treated with control Ig, complete rejection was achieved with both anti-CTLA4 mAbs. In multiple experiments, the two antibodies were equivalent in inducing tumor rejection. In another tumor model, B16 melanoma, both antibodies induced similar tumor growth delay, but complete rejection was not achieved with either antibody (Figure 20).

[0070] Example 7: Antitumor effects are associated with depletion of intratumoral Tregs In vivo immune regulation is achieved by a balance between immune cell activation and immune checkpoints. Specifically, regulatory T cells (Tregs) are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune diseases. Recent studies have shown that the therapeutic efficacy of anti-murine CTLA4 mAbs is influenced by Fc subclass and host Fc receptors, which in turn selectively affect Treg antibody-dependent cellular cytotoxicity within the tumor microenvironment (52, 53). Since differential CTLA4 blocking activity in vivo is unlikely to translate into differences in antitumor activity, we focused on Tregs within the tumor microenvironment in an attempt to establish the mechanism(s) by which antitumor activity occurs. To do this, we sacrificed MC38 tumor-bearing mice before complete rejection (Figure 21) and analyzed the effects of CTLA4 mAbs administered with control Ig, 10D1, or L3D10. h / h The frequency of Tregs was analyzed in knock-in mice. Neither antibody reduced splenic Tregs (Figure 22C), but both reduced Tregs in the tumor microenvironment (Figure 22E). Interestingly, 10D1, but not L3D10, expanded splenic Tregs. The expansion of splenic Tregs by 10D1 recapitulates the clinical finding that ipilimumab increased FOXP3 expression by peripheral blood leukocytes (54). Blockade of B7-CTLA4 interaction does not contribute to Treg depletion, as blocking and non-blocking antibodies were equivalent in depleting Tregs in the tumor microenvironment. 10D1 does not block B7-CTLA4 interaction in vivo, but it does increase CTLA4 expression. h / h Blockade of this interaction is not required for therapeutic efficacy, as it confers therapeutic efficacy in mice and melanoma patients. Furthermore, blocking CTLA4-B7 interaction does not enhance the therapeutic efficacy of the antibody, as two mAbs with dramatically different blocking efficacies exhibit comparable therapeutic efficacy and selective depletion of Tregs in the tumor microenvironment.

[0071] Supporting this observation, anti-human CTLA4 mAb can bind up to 50% of CTLA4 molecules, and neither antibody can block the B7-CTLA4 interaction to achieve upregulation of B7 on dendritic cells. m / h We examined the therapeutic effects of two anti-CTLA4 mAbs in mice (Fig. 16). Again, both antibodies induced rapid rejection of MC38 tumors, although L3D10 was slightly more effective than 10D1 (Fig. 22B). Correspondingly, both antibodies selectively depleted Tregs in the tumor microenvironment (Figs. 22D and 21F). These genetic data further demonstrate the lack of correlation between CTLA4 blockade and local Treg depletion in tumor rejection, thus challenging the commonly held hypothesis that anti-CTLA4 mAbs induce cancer immunity by blocking B7-CTLA4 interactions (10).

[0072] Example 8. Evaluation of the blocking activity of commonly used anti-mouse CTLA4 mAbs 9H10 and 9D9 The concept that CTLA4 is a cell-intrinsic negative regulator of T cell regulation was proposed based on the stimulatory effects of two anti-mouse CTLA4 mAbs, 4F10 and 9H10, both intact and Fab forms (30, 31), but no data have been presented demonstrating that these antibodies block the B7-CTLA4 interaction. More recently, a third anti-mouse CTLA4 mAb, 9D9, was reported to exhibit therapeutic efficacy in tumor-bearing mice, causing local depletion of Tregs in the tumor microenvironment (52). Therefore, we sought to test the ability of all three commercially available anti-mouse CTLA4 mAbs shown to induce tumor rejection to block the B7-CTLA4 interaction in a physiologically relevant context. As an initial test, we used increasing amounts of anti-CTLA4 mAb (up to 2,000-fold molar concentration over CTLA4-Fc) to block the binding of biotinylated CTLA4-Fc to plate-immobilized B7-1 and B7-2. As shown in Figure 23A, anti-mouse CTLA4 mAb 9H10 did not block the B7-1-CTLA4 interaction, even at the highest concentration tested, although moderate blocking was observed when 9D9 was used at very high concentrations. mAb 9D9 effectively blocked the B7-2-CTLA4 interaction, whereas 9H10 failed to do so (Figure 23B). Interestingly, 9D9 exhibited strong binding to soluble CTLA4-Fc, whereas 9H10, despite being more potent than 9D9 in binding to immobilized mouse CTLA4-Fc (Figure 23D), showed poor binding (Figure 23c). Because the failure to observe any blocking activity by 9H10 in this assay may simply reflect poor binding to soluble CTLA4-Fc, we again performed a CTLA4 assay using WT mice (CTLA4 m / mWe measured in vivo blockade of the B7-CTLA4 interaction using upregulation of B7-1 and B7-2 on dendritic cells in mice. As shown in Figures 23E and 23F, 9H10 did not upregulate B7-1 expression on DCs, whereas 9D9 increased B7-1 levels by 15% (P<0.05). Interestingly, 9D9 clearly upregulated B7-2 on DCs, whereas 9H10 failed to do so. Thus, 9H10, the first and most widely studied anti-CTLA4 mAb for tumor immunotherapy, does not block the B7-CTLA4 interaction. Therefore, blockade of the B7-CTLA4 interaction does not contribute to the induction of anti-tumor immunity by anti-mouse CTLA4 mAbs. Because both mAbs exhibited comparable immunotherapeutic efficacy and comparable depletion of Tregs in the tumor microenvironment (52), local depletion of Tregs rather than blockade of B7-CTLA4 interactions provides a unifying explanation for the therapeutic effects of anti-mouse CTLA4 mAbs. Interestingly, 4F10 blocked B7-CTLA4 interactions in vitro but failed to induce upregulation of B7 on DCs in vivo (Figure 24).

[0073] Taken together, we demonstrated that clinically proven therapeutic anti-human CTLA4 mAb (10D1) and two anti-murine CTLA4 mAbs (9H10 and 4F10) confer immunotherapeutic effects under physiologically relevant conditions without blocking the B7-CTLA4 interaction. Furthermore, even in the case of a mAb (L3D10) that could potently block the B7-CTLA4 interaction, such blockade was not required for tumor rejection. Even in the case of antibodies with a 1000-fold difference in blocking the B7-CTLA4 interaction, the therapeutic effect was virtually the same, indicating that such blockade does not contribute to the cancer therapeutic efficacy of anti-CTLA4 mAbs. These data challenge the hypothesis that anti-CTLA4 mAbs confer immunotherapeutic effects through checkpoint blockade (55). By challenging the prevailing hypothesis, our data suggest that the therapeutic efficacy of anti-CTLA4 mAbs cannot be optimized by improving their blocking activity. Of particular interest in this context is that tremelimumab (56), which is effective in blocking the B7-CTLA4 interaction, failed to reach clinical endpoints in a phase III clinical trial (57). On the other hand, by demonstrating a strong correlation between tumor rejection and local Treg depletion and by challenging the involvement of blockade of the B7-CTLA4 interaction in tumor immunity, our study supports the hypothesis that local Treg depletion within the tumor environment is the primary mechanism of action of therapeutic anti-CTLA4 mAbs and thus proposes a new approach to developing next-generation anti-CTLA4 mAbs for cancer immunotherapy.

[0074] Finally, accumulating mouse genetic data suggest that the previous notion (30, 31) that CTLA4 negatively regulates T cell activation and that such regulation is achieved through SHP-2 (58, 59) may need to be reconsidered (60). - / -Although severe autoimmune disease in mice has been used to support the idea that CTLA4 is a cell-intrinsic negative regulator of T cell activation (61, 62), at least three lines of genetic data have since emerged that are inconsistent with this view. First, lineage-specific deletion of the Ctla4 gene in Tregs, but not in effector T cells, is sufficient to recapitulate the autoimmune phenotype observed in mice with germline deletion of the Ctla4 gene (50). These data support the notion that Ctla4 - / - This suggests that the autoimmunity in the mice was not due to the lack of the cell-intrinsic negative regulator CTLA4 in effector T cells. - / - In chimeric mice composed of both T cell types, the coexistence of wild-type T cells prevented the autoimmune phenotype (63). These data also strongly argue that autoimmune disease is not caused by a lack of cell-intrinsic negative regulators. The lack of effect of cell-intrinsic negative regulators is due to the lack of Ctla4 expression during viral infection in chimeric mice. - / - This is also evidenced by the fact that non-preferential proliferation of T cells was observed (64). Third, T cell-specific deletion of Shp2, which was proposed to mediate negative regulation of CTLA4 (58, 59), was found to reduce rather than enhance T cell activation (65). In light of these genetic data reported since CTLA4 was proposed as a negative regulator of T cell activation, our data reported herein call for a reevaluation of CTLA4 checkpoint blockade in cancer immunotherapy.

[0075] Example 9. Chimeric L3D10 demonstrates reduced immune adverse events when used in combination with other immunotherapeutic antibodies Recent clinical trials have demonstrated that combination therapy with anti-PD-1 and anti-CTLA4 mAbs further improves survival in patients with advanced melanoma. However, 55% of patients receiving the combination therapy developed grade 3 and 4 immune-related adverse events (irAEs). Therefore, developing less toxic antibodies is crucial. We developed an in vivo model that reproduces the irAEs associated with combination therapy of anti-CTLA-4 and anti-PD-1 mAbs observed in the clinic. In this model, human CTLA4 gene knock-in mice (CTLA4) were introduced during the perinatal period. h / h ) were treated with high doses of anti-PD-1 and anti-CTLA-4 mAbs. While young mice tolerated the individual mAb treatments, the combination of anti-PD-1 and L3D10 was found to cause severe irAEs, including multiorgan inflammation, anemia, and severe growth retardation, as shown in Figure 25. In contrast, when combined with anti-PD-1, chimeric L3D10 exhibited only mild irAEs, as indicated by normal weight gain.

[0076] To further examine the relative toxicity of chimeric L3D10 compared with 10D1 when administered in combination with anti-PD-1, we performed a CTLA4 h / hWe focused on the pathological effects in the knock-in mice. As shown in Figure 26, 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, in comparison, the weight of mice treated with 10D1 + anti-PD-1 was significantly lower. Based on this, we examined the gross morphology of these mice and found that the uterus, ovaries, bladder, and thymus were significantly smaller in mice treated with 10D1 + PD-1 (Figure 27). Again, the organs of mice treated with L3D10 + anti-PD-1 were comparable to those of the hIgG control. In contrast, hearts isolated from mice treated with 10D1 appeared slightly larger in size and had a distinctly whiter appearance. Therefore, we examined erythropoiesis in the mice and observed a clear difference in mice treated with 10D1 + anti-PD-1 compared to the groups treated with L3D10 + anti-PD-1 or control antibody, which were very similar. As shown in Figure 27A, the bone marrow of mice treated with 10D1 + anti-PD-1 was noticeably white, and isolated blood was almost entirely white (Figure 28b). Accordingly, CD71 and CD119 markers were used to more closely examine cells undergoing different stages of hematopoiesis. Representative FACS profiles are shown in Figure 28C, and summary data are presented in Figure 28D. These data revealed a statistically significant decrease in the number of cells undergoing stage IV development in mice treated with 10D1 + anti-PD-1 (Figure 28D).

[0077] To explore the potential mechanism of anemia in 10D1-treated mice, we investigated whether 10D1+PD-1 treatment induced anti-erythrocyte antibodies. As shown in Figure 29, no anti-erythrocyte antibodies were detected. Therefore, the development of erythrocyte-specific autoantibodies is not involved in the anemia in mice treated with anti-PD-1+10D1.

[0078] To further determine the toxicity of L3D10 versus 10D1 in combination with anti-PD-1, histological analysis was performed on the heart (Figure 30), lungs (Figure 31), salivary glands (Figure 32), and kidneys and liver (Figure 33) after fixation in 10% formalin for at least 24 hours. In each of the tissues examined, mice treated with 10D1 + anti-PD-1 exhibited high levels of T cell infiltration. Toxicity scores based on the severity of inflammation are summarized in Figure 34, showing higher toxicity scores for mice treated with 10D1 + anti-PD-1 compared to L3D10 + anti-PD-1, which had slightly higher scores than the hIgG control mice group.

[0079] Example 10: L3D10 exhibits reduced binding to soluble CTLA4 L3D10 and 10D1 show similar binding patterns to plate-immobilized CTLA4 (Figure 36). We investigated binding to soluble CTLA4 as a possible explanation for the lower toxicity of L3D10 compared to 10D1, particularly the increased T cell infiltration / activation associated with 10D1. We chose to investigate this because the association of CTLA4 polymorphisms with multiple autoimmune diseases is associated with impaired production of soluble CTLA4 (Nature 2003, 423:506-511), and gene silencing of the sCTLA4 isoform increased the incidence of type 1 diabetes in mice (Diabetes 2011, 60:1955-1963). Furthermore, soluble CTLA4 (abatacept and belatacept) are widely used drugs for immunosuppression. In accordance with this data, we observed a significant decrease in L3D10 binding when examining its relative binding to soluble CTLA4 (Figure 37).

[0080] We demonstrated that anti-CTLA-4 mAb induces heterozygous Ctla4, where only 50% of the CTLA-4 molecules can bind to the anti-human CTLA-4 mAb. h / m We demonstrated that CTLA-4 induces potent tumor rejection in mice. To determine whether 50% CTLA-4 binding is sufficient to induce irAEs, we analyzed CTLA-4 h / m Mice were treated with anti-PD-1+10D1. As shown in Figure 35, anti-PD-1+10D1 inhibited Ctla4h / m The study failed to induce weight loss in mice, suggesting that irAEs and cancer immunity can be genetically dissociated.

[0081] In vivo activity demonstrated that the L3D10 antibody retains its antitumor activity but exhibits reduced autoimmune adverse effects observed with other immunotherapeutic antibodies, such as 10D1, suggesting that antitumor activity can be enhanced without exacerbating autoimmune adverse events. Therefore, autoimmune side effects are not a necessary tradeoff for cancer immunity, and these activities can be uncoupled. Characterization of L3D10 demonstrated that its ability to block the interaction of CTLA4 with B7.1 and B7.2 was more effective than that of 10D1, which is associated with differences in the CTLA4-binding sites between the antibodies. Furthermore, L3D10 was fused to a modified human IgG1 Fc domain with mutations that conferred potent ADCC activity, enhancing the antibody's therapeutic efficacy. Further characterization demonstrated that L3D10 and 10D1 bind to immobilized CTLA4 with similar binding profiles. However, L3D10 exhibits much lower binding affinity for soluble CTLA4 than 10D1. Taken together, our data demonstrate that antibody L3D10 has great potential for clinical use in the treatment of cancer patients with less severe adverse events.

[0082] Example 11. Humanization of L3D10 The humanization process begins by generating a 3D structure of a homologous model antibody and profiling the parent antibody based on structural modeling. Acceptor frameworks were identified based on overall sequence identity across the framework, matching interface positions, canonical positions of similarly classified CDRs, and the presence of N-glycosylation sites that needed to be removed. One light chain (LC) and one heavy chain (HC) framework were selected for humanization design.

[0083] Humanized antibodies were designed by creating multiple hybrid sequences fusing selected portions of the parent antibody sequence with human framework sequences (including grafting CDR sequences into the acceptor framework). The predicted CDR sequences of the parent antibody L3D10 are provided as SEQ ID NOS: 21-26, as shown in Table 1A below. [Table 1A]

[0084] Using the 3D model, these humanized sequences were systematically analyzed visually and by computer modeling to isolate sequences that would be most likely to retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibody while retaining the original antibody properties.

[0085] Based on the selected acceptor framework, three humanized light chains (LC1, LC2, and LC3) and three humanized heavy chains (HC1, HC2, and HC3) were designed. Each of the three HC or three LC sequences was derived from the same germline and had different backmutations in the murine parental sequence, as shown in Figure 38. The amino acid sequences of the humanized variable regions and their optimized encoding nucleotide sequences are set forth in SEQ ID NOS: 9-20. The CDR2 sequences of both the humanized heavy and light chains contain amino acid changes relative to the parent L3D10 antibody sequence and are set forth in SEQ ID NOS: 33-38, as shown in Table 1B below. [Table 1B]

[0086] The humanized light and heavy chains can then be combined to create a variant fully humanized antibody. All possible combinations of humanized light and heavy chains were tested for their expression levels and antigen affinity to identify antibodies with similar performance to the parent antibody.

[0087] We used a new tool (24) to calculate the humanness score of monoclonal antibodies. This score represents how human-like the antibody variable region sequence is and is an important factor when humanizing antibodies. The humanness scores of the parent and humanized antibodies are shown in Tables 2 and 3 below. According to our method, for the heavy chain, a score of 79 or higher indicates human-like appearance, and for the light chain, a score of 86 or higher indicates human-like appearance. [Table 2] [Table 3]

[0088] Full-length antibody genes were constructed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into an expression vector that already contained a human Fc domain; for the heavy chain, a hIgG1 (M252Y, S254T, T256E, S298A, E333A, K334A) backbone was used. Additionally, for comparison, chimeric parent heavy and light chain variable regions were constructed as full-length chimeric chains using the same backbone Fc sequence.

[0089] All nine humanized antibodies were produced in small-scale production runs of 0.01 liters. The chimeric parent antibody was also scaled up for direct comparison. Antibodies were produced by transfecting the indicated heavy and light chain plasmids into suspension HEK293 cells using chemically defined media in the absence of serum. Total antibodies were purified in conditioned media using MabSelect SuRe Protein A medium (GE Healthcare). The 10 antibodies tested are listed in Table 4 below. [Table 4]

[0090] The affinity of nine humanized antibody combinations and the chimeric parent antibody (huCTLA4) for antigen was evaluated using Octet. Multiple-concentration kinetic experiments were performed on an Octet Red96 system (ForteBio). Anti-hIgG Fc biosensors (ForteBio, no. 18-5064) were hydrated in sample diluent (0.1% BSA and 0.02% Tween 20 in PBS) and preconditioned in pH 1.7 glycine. Starting at 600 nM, antigen was diluted using seven 2-fold serial dilutions in sample diluent. All antibodies were diluted to 10 μg / mL in sample diluent and then immobilized on the anti-hIgG Fc biosensor for 120 seconds. After establishing a baseline in sample diluent for 60 seconds, the biosensor was transferred to wells containing a series of antigen concentrations to measure association. Association with each protein of interest in sample diluent was observed for 120 seconds, and dissociation was observed for 180 seconds. Binding affinity was characterized by fitting the kinetic sensorgrams to a monovalent binding model (1:1 binding). Full kinetic measurements are summarized in Table 5 below. [Table 5]

[0091] Example 12. Antitumor activity of humanized anti-CTLA4 antibodies Based on relative binding affinity and humanness scores, three antibodies were selected for further evaluation. PP4631 - High affinity and good expression PP4637 - High affinity and good expression PP4638 - slightly lower affinity but highest humanization score Material for each of these antibodies was generated by transient production in HEK293 cells at a 0.1 liter scale followed by purification with Protein A. The binding affinity of the purified antibodies was confirmed by Octet analysis, as shown in Table 6 below. [Table 6]

[0092] The anti-tumor activity of these three humanized antibodies was evaluated in comparison with 10D1 and the chimeric L3D10 antibody using the syngeneic MC38 mouse tumor model of human CTLA4-knockin mice described above in Example 5. Figure 39A shows the treatment schedule for the in vivo experiment: mice were administered a total of four doses of antibody every three days starting on day 7 post-inoculation. As shown in Figure 39B, all humanized antibodies completely eradicated tumors, comparable to 10D1.

[0093] In a separate experiment, heterozygous Ctla4 as described in Example 5 h / m The antitumor activity of humanized antibodies PP4631 and PP4637 was evaluated in comparison with 10D1 and the chimeric L3D10 antibody at two different doses using a syngeneic MC38 mouse tumor model in mice (Figure 14). As shown in Figure 40, all mAbs were indistinguishable when used at 30 mcg / mouse / injection (1.5 mg / kg), whereas PP4637 was more effective at 10 mcg / mouse / injection (0.5 mg / kg), and PP4631 and 10D1 showed comparable activity.

[0094] The anti-tumor activity of the humanized antibodies compared to 10D1 and the chimeric L3D10 antibody was also determined using a syngeneic B16-F1 melanoma mouse tumor model in human CTLA4-knockin mice, as shown in Figure 41. Mice were administered a total of three doses of antibody every three days, starting two days after inoculation. As shown in Figure 41, L3D10 and the humanized antibody delayed tumor growth comparable to 10D1.

[0095] Example 13. Humanized clones of L3D10 maintain a superior safety profile to 10D1 To test whether the excellent safety profile of L3D10 could be maintained after humanization, PP4631 and PP4637 were compared with 10D1 for their adverse effects when used in combination with anti-PD-1. As shown in Figure 42, both PP4631 and PP4637 were less toxic than 10D1 when used in combination with anti-PD-1.

[0096] Consistent with the erythropoiesis deficiency described in Figure 28, complete blood counts (CBCs) showed that mice treated with 10D1 + anti-PD-1 were anemic, whereas mice treated with anti-PD-1 + PP4631 and anti-PD-1 + PP4637 had near-normal CBC profiles, as shown in Figure 43. Furthermore, analysis of T cell profiles in PBLs revealed robust systemic activation of both CD4 and CD8 T cells in mice treated with 10D1 + anti-PD-1, but not in mice treated with anti-PD-1 + PP4631 or anti-PD-1 + PP4637 (Figure 44), further supporting the notion that L3D10-based anti-CTLA-4 mAbs do not cause systemic T cell activation.

[0097] Example 14. Binding characteristics of humanized anti-CTLA4 antibodies To confirm that the humanized antibodies retained their CTLA4-binding properties, binding to immobilized CTLA4 and plate-bound CTLA4 was examined. As shown in Figure 45, humanization did not affect binding to immobilized CTLA4, and all three humanized antibodies exhibited binding similar to the parent chimeric L3D10 antibody. However, as shown in Figure 46, humanization further reduces the binding of L3D10 to soluble CTLA4. Based on the reduced binding to soluble CTLA4, the three humanized antibodies are expected to induce equivalent tumor rejection with even fewer autoimmune side effects than L3D10.

[0098] We demonstrated that chimeric L3D10 has 1000-fold greater blocking activity than 10D1. This raises the intriguing possibility that blocking the B7-CTLA-4 interaction may explain the lack of irAEs. As shown in Figures 47 and 48, neither PP4631 nor PP4637 block the B7-CTL-A4 interaction in vitro or in vivo. The fact that PP4631 and PP4637 show reduced irAEs further supports the notion that blocking the B7-CTLA-4 interaction does not contribute to the improved safety of L3D10.

[0099] Given the proposed role of CTLA-4 in protecting against autoimmune disease, we proposed reduced binding to soluble CTLA-4 as the mechanism underlying the improved safety profile. To test this hypothesis, growth (weight gain) among female mice administered anti-PD-1 plus anti-CTLA-4 mAb during the perinatal period was used as a baseline indicator of irAEs. As shown in Figure 42, a significant decrease in weight gain was observed in mice administered both 10D1 and anti-PD-1, whereas mice administered PP4637 plus anti-PD-1 had the fewest irAEs, followed by PP4631 and then L3D10. The strict correlation with reduced binding to sCTLA-4 is consistent with our primary hypothesis.

[0100] Example 15. Processability evaluation of humanized anti-CTLA4 antibodies To evaluate the feasibility of developing and manufacturing three different humanized antibodies, multiple analytical methods were performed to characterize the different antibodies. [Table A]

[0101] As an initial evaluation, the predicted molecular weights and isoelectric points of the three lead candidate antibodies were calculated based on their amino acid sequences. As shown in Table 7, all antibodies were quite similar, although the antibodies had somewhat lower PIs. [Table 7]

[0102] Product yield assessment To evaluate the productivity 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 using serum-free medium. After 6 days, the supernatant was collected and the antibodies were purified by one-step Protein A chromatography. As can be seen in Table 8 below, antibodies PP4631 and PP4637 showed similar protein yields, while antibody PP4638 was produced at a much lower relative yield. [Table 8]

[0103] To assess the purity of the transiently expressed antibodies, samples were analyzed by reducing and non-reducing SDS-PAGE. As shown in Figure 50, samples from all three antibodies produced gel bands indicative of antibody molecules, and after purification with Protein A, the samples were relatively pure.

[0104] Size exclusion chromatography To further investigate the purity and aggregation of the different antibodies after transient expression, size-exclusion chromatography of the purified proteins was performed. Briefly, 50 μg of filtered (using a 0.22 μm filter) sample was used for SE-HPLC separation using a TOSOH G3000 SWxl 5 μm column. PBS (pH 7.4) was used as the mobile phase. As shown in Table 9 below, all humanized antibodies showed >90% purity after purification with Protein A. Antibodies PP4631 and PP4637 showed similarly low levels of higher molecular weight (MW) aggregates and degradation, with the antibody sample exhibiting most of the protein within the main peak. In contrast, antibody PP4638 showed higher levels of aggregation and some degradation. The SE-HPLC chromatograms are shown in Figure 51. [Table 9]

[0105] Capillary electrophoresis (CE) Capillary electrophoresis was used to quantify the amount of protein in the peak bands under both reducing and non-reducing conditions, as well as the amount of unglycosylated heavy chain protein. Briefly, 100 μg of sample was diluted in CE-SDS sample buffer with 2 μL of a 10 kDa standard protein and either iodoacetamide (non-reducing conditions) or β-mercaptoethanol (reducing conditions). The sample was then treated for 10 minutes at 70°C. A PA-800, 50 μm ID bare fused silica capillary was used for separation: running length 20.2 cm, separation voltage 15 kV, OD220 As shown in Table 10 below, all three proteins showed a high level of purity, consistent with SDS-PAGE, and all were heavily glycosylated. The CE-SDS chromatograms are shown in Figure 52. [Table 10]

[0106] Deamidation: Capillary Isoelectric Focusing (cIEF) and Liquid Chromatography-Mass Spectrometry (LC-MS) The antibody was compared with and without high pH stress treatment for two different periods (5 h and 12.5 h) followed by cIEF and LC-MS analysis to determine the level of protein deamidation under high pH stress.

[0107] The charge isoform profile and isoelectric point of the different antibodies were determined by capillary isoelectric focusing (cIEF). Briefly, samples were buffer exchanged into 20 mM Tris (pH 8.0), and then 100 μg of sample protein was mixed with ampholytes, methylcellulose, and markers PI 7.05 and PI 9.77. Analysis was performed using an iCE3™: 100 μm ID capillary, 1.5 kV + 3 kV, OD 280 Detected by . For deamidation stress treatment, samples were treated with 500 mM NaHCO3 for 5 or 12.5 hours and then examined by cIEF and LC-MS. The results of the analysis are shown in Table 11 below, and the LC-MS graph is shown in Figure 53. All three antibodies show the expected increase in the amount of deamidated species under stress conditions, with a corresponding decline in the main peak. As predicted from the amino acid sequence, the pI of antibody PP4637 is slightly lower than that of PP4631 and PP4638 (Table 7), and the observed higher pI compared to the predicted pI is likely indicative of glycosylation. [Table 11]

[0108] Differential Scanning Calorimetry (DSC) Thermal Analysis To determine the thermal stability and melting temperatures of the different antibodies, they were subjected to differential scanning calorimetry (DSC) thermal analysis. Briefly, 2 mg / mL samples in PBS (pH 7.4) were subjected to a temperature ramp from 15°C to 105°C at a rate of 1°C / min. The Cp change with temperature was monitored for both the sample and the buffer (as background). Background-subtracted Cp vs. temperature curves were obtained, with peaks indicating the Tm of the analytes. As shown in Table 12 below, all three antibodies exhibited similarly high melting temperatures. The DSC curves of the three antibodies are shown in Figure 54. [Table 12]

[0109] Oxidation: Peptide Mapping Oxidative modification of the humanized antibody was assessed by peptide mapping using LC-MS with or without oxidative stress. Samples were denatured at 65°C in the presence of 6M GnCl and 5mM β-ME, then acetylated with iodoacetamide. The processed samples were then digested with trypsin (Promega, sequencing grade) at 55°C. The digested mixture was separated on a C18 reversed-phase LC column (ACQUITY UPLC BEH130 C18, 2.1 x 100 mm, 1.7 μm) and analyzed by mass spectrometry (Waters XEVO-G2S QTOF) using Masslynx and Biophatmlynx analytical tools. For oxidative stress analysis, samples were treated with 0.05% or 0.1% HO for 1 hour and then examined by LC-MS. The results are shown in Tables 13–16 below. [Table 13] [Table 14] [Table 15] [Table 16]

[0110] Binding specificity The binding specificity of the different antibodies was determined by assessing their ability to detect nonspecific binding to two different cell lines (CHO and HEK293) that do not express CTLA4 at two different concentrations compared to 10D1. Briefly, 100 μg / mL or 20 μg / mL of sample (or reference mAb) in PBS was added to 3 × 10e 6 The cells were incubated with 1000 cells / ml (CHO or HEK293). FITC-labeled rabbit anti-human IgG antibody (Boster, Wuhuan, China) was used for detection, and the binding of the target mAb to the cells was measured by FACS. As shown in Table 17 below, antibodies PP4631 and PP4637 showed very low binding and good specificity, while antibody PP4638 showed nonspecific binding activity to the control cell line. [Table 17]

[0111] Example 16. Epitope mapping of L3D10 antibody and humanized antibody To map the CTLA-4 binding epitopes of the L3D10 parent antibody and the humanized variants PP4631 and PP4637, we took advantage of the fact that mouse and human CTLA-4 proteins are cross-reactive with B7-1 but not with anti-CTLA-4 antibodies. To do so, we designed a number of mutants of the human CTLA-4Fc protein in which clusters of amino acids 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, replacing key residues in the antibody binding epitope with mouse amino acids should abolish binding of the anti-human CTLA-4 antibody. DNA vectors encoding 11 CTLA-4Fc mutant proteins (M1 to M11) (SEQ ID NOs: 40 to 50) based on the wild-type human CTLA-4Fc sequence were constructed, and the proteins were produced by transient transfection into HEK293 cells at a 0.01 mL scale, followed by single-step purification using Protein A. Binding of anti-CTLA-4 antibodies to CTLA-4Fc protein was performed by ELISA. Plates were coated with 1 μg / mL of CTLA-4Fc protein, and then biotinylated antibodies or B7-1Fc fusion proteins were used in the soluble phase in binding assays. The amount of bound protein was measured using horseradish peroxidase (HRP)-conjugated streptavidin. Anti-human CTLA-4 antibodies did not cross-react with mouse CTLA-4, likely reflecting amino acid sequence differences between human and mouse CTLA-4 in the extracellular domain. Figure 55 shows an alignment of the human, macaque, and mouse CTLA-4 extracellular domains, highlighting sequence conservation between human and macaque while revealing many differences between the mouse and primate sequences. Due to the conservation of the MYPPPY-binding motif, mouse and human CTLA-4 proteins are cross-reactive with B7-1 (72). To map the binding epitope of anti-human CTLA-4 antibodies, a number of non-redundant CTLA-4Fc muteins were generated that incorporated a cluster of mouse-specific amino acids into the human CTLA-4 sequence. The amino acids incorporated into each of the 11 mutants are shown in Figure 55, and the amino acid sequences of the 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 of the mutations appear to affect protein expression, as indicated by the yield compared to the WT human CTLA-4Fc protein. [Table 18]

[0112] The ability of the chimeric L3D10 antibody and the humanized antibodies PP4631 and PP4637 to bind to immobilized CTLA-4 Fc mutant constructs was then determined by ELISA: plates were coated with the CTLA-4 mutant constructs, biotinylated anti-CTLA-4 antibody or B7-1 Ig control protein was added, and binding was measured using HRP-conjugated streptavidin. The results of the binding assays are shown in Tables 19-22. As expected, all four binding proteins showed good dose-dependent binding to the WT CTLA-4 Fc protein. However, the mutations introduced into the M9 and M10 proteins appeared to alter the overall structure, and these mutants were unable to bind B7-1 Fc. The mutations introduced into M2 and M4 also partially altered the conformation of CTLA-4, as suggested by the reduced binding compared to the WT protein. Consistent with this notion, all four of these mutants (M2, M4, M9, and M10) were expressed in much lower yields (Table 18). In contrast, using binding to WT CTLA-4 Fc protein and B7-1 Fc protein as references, M11 clearly stands out as a well-expressed protein, binding efficiently to B7-1 Fc but failing to bind to the two humanized anti-CTLA-4 antibodies. Binding to the native L3D10 is also reduced approximately 100-fold (Table 20). As expected, mutations affecting the overall conformation also affected binding to the anti-CTLA-4 antibodies. [Table 19] [Table 20] [Table 21] [Table 22] [Table 23]

[0113] Because L3D10 retained significant binding to M11, we investigated whether its binding was specific. Plates were coated with human CTLA4-Fc (hCTLA4Fc), mouse CTLA4-Fc (mCTLA4-Fc), control IgG1-Fc, or all mutant hCTLA4-Fc, and their binding to B7-1Fc was measured along with L3D10, PP4631, and PP4637. Overall data are presented in Table 23. As shown in Figure 57, biotinylated B7-1 binds equally well to hCTLA-4, mCTLA-4, and M11. The specificity of the assay is demonstrated by the lack of binding to IgG1-Fc. Interestingly, while binding of L3D10 to M11 was stronger than that to IgG1-Fc and mCTLA4-Fc, the significant binding to IgG1-Fc suggests that the chimeric antibody's binding to M11 may be nonspecific. In contrast, none of the humanized antibodies bind to M11, mCTLA-4, or the IgG1-Fc control. These data indicate that the mutations introduced into M11 selectively abrogated the binding of L3D10, PP4631, and PP4637 to CTLA-4.

[0114] Using the known complex structure,133, the CTLA-4 epitope was mapped in the 3D structure. As shown in Figure 58, the epitopes recognized by these mAbs were localized to the region covered by B7-1. Therefore, the binding of L3D10, PP4631, and PP4637 to CTLA-4 appears to be mutually exclusive to B7-1 binding. The poor blocking of PP4631 and PP4637 is due to lower avidity rather than unique binding domains.

[0115] Taking advantage of the fact that mouse and human CTLA-4 proteins are cross-reactive with B7-1, but anti-human CTLA-4 antibodies do not cross-react with mouse CTLA-4, we were able to map the binding epitope of the L3D10-derived antibody by ELISA. Using multiple mutants of the human CTLA-4Fc protein in which clusters of amino acids from human CTLA-4 were replaced with amino acids from mouse CTLA-4, we clearly demonstrated that substitution of the four amino acids immediately following the known B7-1-binding domain of CTLA-4 abolished most of the antibody's dose-dependent binding. The fact that the binding epitope is located directly adjacent to the B7-1-binding domain correlates well with the demonstrated ability of the L3D10 antibody to block B7-CTLA-4 interactions both in vitro and in vivo. Because soluble CTLA4 is generated by fusion of the C-terminal amino acids of the extracellular IgV domain to the intracellular domain, it is tempting to speculate that antibodies that bind to polymorphic C-terminal domain residues (only 18 amino acids from the C terminus) are likely to lose reactivity to soluble CTLA-4 in which the large intracellular domain has been fused to the C terminus of the extracellular domain.

[0116] To further explore the binding domain of the anti-CTLA4 antibodies, six additional mutant CTLA4-Fc fusion proteins (SEQ ID NOs: 51-56), designated M12-M17, were designed (Figure 59) and used to compare binding to the anti-CTLA4 antibodies 10D1 (Figure 60A), PP4631 (Figure 60B), and PP4637 (Figure 60C). As shown in Figure 60, the Y 103 L 104 I 106 Mutations in M11 at 1000 bp abrogated binding to 10D1, PP4631, and PP4637, suggesting that the binding site for 10D1, PP4631, and PP4637 is at residue Y. 103 L 104 I 106 Importantly, a further mutation within A29>Y is 103 L 104 I 106These data suggest that mutations at position A in CTLA4 restore binding of CTLA-4 to PP4631 and PP4637. 29 is important for binding of antibodies PP4631 and PP4637, but not for 10D1.

[0117] Example 17. Anti-CTLA-4 mAb synergizes with anti-4-1BB in inducing tumor rejection Studies in animal models have suggested that antitumor responses elicited by anti-CTLA-4 monoclonal antibodies (mAbs) are due, at least in part, to antigen-specific T cell responses against normal "self" differentiation antigens (73, 74). The tendency of anti-CTLA-4 antibodies to exacerbate autoimmune disease has been well documented in mice (75-78). This view has been further supported by more recent human clinical trials, where patients developed severe autoimmune symptoms that required discontinuation of treatment, proving a significant limitation (79). On the other hand, the cancer therapeutic anti-4-1BB mAb has been shown to suppress the development of autoimmune disease in lupus-prone mice (24, 25).

[0118] The fact that anti-4-1BB mAb can both stimulate antitumor responses and alleviate autoimmune symptoms raises the intriguing possibility that combining this antibody with anti-CTLA-4 mAb might result in cancer rejection without inducing autoimmunity. In this study, anti-CTLA-4 and anti-4-1BB were combined to induce the rejection of large, established tumors.

[0119] Combined effect of anti-mouse CTLA-4 and anti-mouse 4-1BB antibodies in inducing CD8 T cell-mediated tumor rejection We investigated the antitumor effects of combined treatment with anti-mouse-4-1BB and anti-mouse-CTLA-4 mAbs in two models: one with minimal disease and one with large, established tumors. C57BL / 6 mice were challenged with subcutaneous inoculations of MC38 colon cancer cells. Tumor-challenged mice were injected with antibody at different time points after tumor cell inoculation, and tumor size and incidence were monitored by physical examination.

[0120] In the minimal disease model, mice were treated with hamster IgG + rat IgG, anti-4-1BB + hamster IgG (anti-4-1BB alone group), anti-CTLA-4 + rat IgG (anti-CTLA-4 alone group), or anti-4-1BB in combination with anti-CTLA-4 starting 48 hours after tumor cell inoculation. The antibodies were administered intraperitoneally (ip) on days 2, 9, and 16. Treatment with either anti-4-1BB or anti-CTLA-4 mAb alone resulted in tumor growth delay in one of five mice in each group that rejected the tumor, and four of five mice treated with both anti-CTLA-4 and anti-4-1BB mAb were tumor-free at the end of the experiment. Figure 61A shows tumor growth measurements for each mouse. A linear random-effects model was fitted to the data to compare growth rates between groups. Combination therapy reduced the daily increase in tumor size by 4.6 mm compared to anti-CTLA-4 alone. 2 / day (p=0.0094). Furthermore, the combination therapy significantly reduced proliferation by 8.4 mm compared to anti-4-1BB alone. 2 In addition to growth rate, actual tumor size was compared between treatment groups at 6 weeks after initial tumor challenge. The mean tumor size at 6 weeks was 137.8 mm for anti-CTLA-4 (137.8 mm). 2 , p=0.0251) or anti-4-1BB (287.6, p=0.0006) separately. 2 Thus, in the setting of minimal tumor burden, the combination of anti-4-1BB and anti-CTLA-4 mAb results in a significant delay in tumor growth superior to anti-4-1BB or anti-CTLA-4 administered separately.

[0121] To determine whether the antitumor effects of combined mAb treatment on small tumor burdens could be extended to therapeutic use on larger tumor burdens, mice bearing established tumors were treated with the antibodies. Wild-type C57BL / 6 mice were challenged with subcutaneous inoculation of MC38 colon carcinoma cells. Tumors were allowed to grow for 14 days, at which point mice with established tumors (usually >7 mm in diameter) were selected and randomly divided into four treatment groups: hamster IgG + rat IgG, anti-4-1BB + hamster IgG, anti-CTLA-4 + rat IgG, and anti-4-1BB mAb in combination with anti-CTLA-4 mAb. Antibodies were administered intraperitoneally on days 14, 21, and 28 after tumor challenge. As shown in Figure 61B, treatment with anti-CTLA-4 mAb did not interfere with tumor growth compared to control IgG treatment, although rejection was observed in one of eight mice in the group. Treatment with anti-4-1BB mAb slightly slowed tumor growth, but only one of eight mice rejected the tumor. In contrast, combination therapy with both anti-CTLA-4 and anti-4-1BB mAb resulted in tumor eradication in seven of eight mice and prevented further tumor growth in the remaining mice. Growth rates between groups were compared by fitting a linear random-effects model to the data, as described above. Combination therapy reduced the daily increase in tumor size by 10.6 mm compared to anti-CTLA-4 alone. 2 / day (p<0.0001). Furthermore, the combination therapy significantly reduced proliferation by 6.2 mm compared to anti-4-1BB alone. 2 / day significantly reduced tumor growth (p=0.0002). In addition to growth rate, actual tumor size was compared between treatment groups at week 8 after the initial tumor challenge. Estimated mean tumor size at week 8 was 404.9 mm for anti-CTLA-4 (404.9 mm). 2 , 95%CI: 285.4, 524.4mm 2 ;p<0.0001) or anti-4-1BB (228.4mm 2 , 95%CI:200.4, 689.9mm 2 ;The combined treatment mice had significantly smaller tumors (-1.7 mm) compared to mice receiving either drug separately (p=0.0004). 2 , 95%CI:-10.8, 7.5mm 2 Therefore, the combined mAb appears to significantly delay tumor growth compared with anti-CTLA-4 or anti-4-1BB alone, even at higher tumor burdens.

[0122] MC38 is known to form liver metastases 80 To evaluate the effect of therapeutic antibodies on liver metastasis, all mice included in the study were analyzed for liver metastasis by histological examination. As shown in Table 24, approximately 60% of mice treated with control Ig had micrometastases in the liver. Treatment with either anti-CTLA-4 or anti-4-1BB antibody alone slightly reduced the rate of metastasis, but the reduction did not reach statistical significance. Of note, only 1 / 22 mice in the group treated with both antibodies had liver metastases. Using a logistic regression model, we found that the probability of liver metastasis in mice receiving anti-4-1BB alone was approximately 4.7-fold higher than that in mice receiving both anti-4-1BB and anti-CTLA-4 (95% CI: 1.6, 13.7; p=0.0050). Similarly, mice receiving anti-CTLA-4 alone had a 3.6-fold increased likelihood of liver metastasis compared to mice receiving both treatments (95% CI: 1.3, 10.2; p=0.0174). Thus, combination therapy significantly reduces MC38-induced liver metastasis compared to treatment with either antibody alone. [Table 24]

[0123] To determine which immune cell subsets contributed to the antitumor effects induced by the combined mAb treatment, we depleted major lymphocyte subsets with monoclonal antibodies. MC38 tumor cells were injected subcutaneously. After tumors became palpable, tumor-bearing mice were divided into four groups. Each group received a series of intraperitoneal antibody injections to deplete different subsets of immune cells (including non-depletion with normal rat IgG, depletion of CD4 T cells with anti-CD4 mAb (GK1.5), depletion of CD8 T cells with anti-CD8 mAb (2.4.3), and depletion of NK cells with anti-NK1.1 mAb (PK136)). In addition, all mice in all groups were treated with anti-CTLA-4 plus anti-4-1BB mAb once a week for 3 weeks. Sufficient immune cell depletion was assessed by flow cytometry of peripheral blood collected from mice immediately before the completion of the experiment (data not shown). As expected, mice that were not immune cell depleted responded to treatment with anti-CTLA-4 in combination with anti-4-1BB mAb (Figure 62). Similarly, depletion of NK cells and CD4 T cells did not affect the anti-tumor activity of anti-CTLA-4 + anti-4-1BB mAb combination therapy. However, depletion of CD8 T cells suppressed the anti-tumor activity of the combined antibody therapy. At day 28, the estimated mean tumor size (92.3 mm) in CD8 T cell-depleted mice was 92.3 mm. 2 , 95%CI:64.5, 120.1mm 2 ) was significantly larger than the mean tumor size (28.7 mm) in mice that were not immune cell depleted. 2 , 95%CI:-17.1, 74.4mm 2 ), CD4 T cell-depleted mice (16.7 mm 2 , 95%CI:1.0, 32.4mm 2 ), and NK cell-depleted mice (9.3 mm 2 , 95%CI:-8.3, 26.9mm 2 These data indicate that the tumor eradication effect of anti-CTLA-4 and anti-4-1BB mAb treatment is CD8 T cell dependent.

[0124] Anti-4-1BB antibody reduced the antibody response to heterologous anti-CTLA-4 antibody. 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 effect of anti-4-1BB antibody on the host response to anti-CTLA-4 antibody. As shown in Figure 63, very little, if any, anti-antibody response was detected in mice treated with either control IgG or anti-4-1BB. Consistent with the ability of anti-CTLA-4 mAb to promote CD4 T cell responses, 82 Mice treated with anti-CTLA-4 plus rat IgG exhibited a strong host antibody response to the administered 4F10 antibody and rat IgG (Figures 63A-B). This response was reduced by more than 30-fold when anti-4-1BB was co-administered with anti-CTLA-4 mAb. These data suggest that anti-4-1BB antibodies can potentially increase the duration of other co-administered therapeutic proteins by reducing the host response to the therapeutic agent.

[0125] In human CTLA-4 knock-in mice, the combination of anti-mouse 4-1BB and anti-human CTLA-4 antibodies reduced tumor rejection and sustained cancer immunity.

[0126] Because anti-4-1BB reduces the production of antibodies against anti-CTLA-4 antibodies, an interesting question is whether the enhanced tumor rejection by anti-4-1BB is simply due to its effect in suppressing antibody responses. These human CTLA4 gene knock-in mice allowed us to test whether the anti-tumor effect of anti-human CTLA4 antibodies could be enhanced by anti-4-1BB antibodies. As shown in Figure 64A, both anti-human CTLA-4 (L3D10) and anti-4-1BB antibodies (2A) alone delayed tumor growth, but the combination of the two antibodies resulted in the most significant tumor rejection. In the groups treated with anti-CTLA-4, 4-1BB, or both antibodies, respectively, 1 / 7, 2 / 7, and 5 / 7 mice did not develop any tumors, whereas all mice in the untreated group developed tumors. Because anti-human CTLA-4 antibodies are of murine origin, the effect of 4-1BB antibodies cannot be attributed to the suppression of antibodies against the therapeutic anti-CTLA-4 antibodies. Furthermore, our data also demonstrated that the superior efficacy of the combination therapy is likely applicable to immunotherapy based on anti-human CTLA-4 antibodies.

[0127] To test whether mice treated with the two antibodies were immune to further tumor cell challenge, the mice were challenged with tumor cells 110 days after the first tumor cell challenge. As shown in Figure 64B, all five mice treated with the two antibodies that initially rejected tumor cells remained tumor-free, whereas control naive mice showed progressive tumor growth. Therefore, the combination therapy also induced persistent immunity against cancer cells.

[0128] One of the obstacles to protein-based immunotherapy is host immunity to the therapeutic protein. In the case of antibodies, the host can mount antibodies against heterotypic, allotypic, and idiotypic epitopes. 81While xenotypic responses can be eliminated by full humanization, other anti-antibody responses require special consideration. In the case of anti-CTLA-4 antibodies, the obstacle is more obvious because they are themselves adjuvants. Previous studies by Mittler et al. demonstrated significant suppression of T cell-dependent humoral immune responses. 83 Our data show that coadministration of anti-4-1BB antibody reduces the host response to anti-CTLA-4 antibody, suggesting another benefit of combination therapy with anti-CTLA-4 and anti-4-1BB antibodies. Taken together, our data indicate that combination therapy with anti-CTLA-4 and anti-4-1BB antibodies offers three major advantages: enhanced efficacy in cancer immunity, reciprocal suppression of autoimmune side effects, and improved anti-antibody responses.

[0129] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover in general any variations, uses, or adaptations of the invention which comply with the principles of the invention and include such departures from the present disclosure as are known or customarily practiced in the art to which this invention pertains, and which may apply to the essential characteristics described hereinabove.

[0130] Cited references 1. Townsend ARM, Tothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. "The epitope of influenza nucleoprotein recognized by cytotoxic lymphocytes can be defined with short synthetic peptides." Cell. 1986;44:959-68. 2. Zinkernagel RM, Doherty PC. “Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system.” Nature. 1974;248:701-702. 3. Lafferty KJ, Prowse SJ, Simeonovic CJ, Warren HS. “Immunobiology of tissue transplantation: a return to the passenger leukocyte concept.” Annu Rev Immunol. 1983;1:143-73. 4. Liu Y, Linsley PS. “Costimulation of T-cell growth.” Curr Opin Immunol. 1992;4(3):265-70 5. Schwartz RH. “Costimulation of T lymphocytes: the role of CD28, CTLA4, and B7 / BB1 in interleukin-2 production and immunotherapy.” Cell. 1992;71(7):1065-8 6. Freeman GJ, Freedman AS, Segil JM, Lee G, Whitman JF, Nadler LM. "B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells." J Immunol. 1989;143(8):2714-22. 7. Freeman GJ, Gribben JG, Boussiotis VA, Ng JW, Restivo VA, Jr, Lombard LA, et al., “Cloning of B7-2: a CTLA4 counter-receptor that costimulates human T cell proliferation” [see comments] Science. 1993;262(5135):909-11. 8. Hathcock KS, Laszlo G, Dickler HB, Bradshaw J, Linsley P, Hodes RJ. "Identification of an alternative CTLA4 ligand costimulatory for T cell activation [see comments]." Science. 1993;262(5135):905-7. 9. Wu Y, Guo Y, Liu Y. “A major costimulatory molecule on antigen-presenting cells, CTLA4 ligand A, is distinct from B7.” J Exp Med. 1993;178(5):1789-93 10. Leach DR, Krummel MF, Allison JP. “Enhancement of antitumor immunity by CTLA4 blockade” [see comments] Science. 1996;271(5256):1734-6 11. Linsley PS, Brady W, Urnes M, Grosmaire LS, Damle NK, Ledbetter JA. “CTLA4 is a second receptor for the B cell activation antigen B7.” J Exp Med. 1991;174(3):561-912. Linsley PS, Clark EA, Ledbetter JA. “T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7 / BB-1B.” Proc Natl Acad Sci USA.1990;87(13):5031-5 13. Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, et al. “Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients.” Proc Natl Acad Sci USA. 2003;100(8):4712-7. PubMed PMID: 12682289 14. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB et al. “Improved survival with ipilimumab in patients with metastatic melanoma.” N Engl J Med. 2010;363(8):711-23. Epub 2010 / 06 / 08. doi:10.1056 / NEJMoa1003466. PubMed PMID: 20525992; PubMed Central PMCID: PMC3549297 15. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. “Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma.” N Engl J Med. 2015;373(1):23-34. Epub 2015 / 06 / 02. doi:10.1056 / NEJMoa1504030. PubMed PMID:26027431 16. Ribas A, Hodi FS, Callahan Callahan M, Konto C, Wolchok J. "Hepatotoxicity with combination of vemurafenib and ipilimumab." N Engl J Med. 2013;368(14):1365-6. Epub 2013 / 04 / 05. doi:10.1056 / NEJMc1302338. PubMed PMID:23550685 17. Delyon J, Mateus C, Lambert T. “Hemophilia A induced by ipilimumab.” N Engl J Med. 2011;365(18):1747-8. Epub 2011 / 11 / 04. doi:10.1056 / NEJMc1110923. PubMed PMID: 22047582 18. Fadel F, El Karoui K, Knebelmann B. “Anti-CTLA4 antibody-induced lupus nephritis.” N Engl J Med. 2009;361(2):211-2. Epub 2009 / 07 / 10. doi:10.1056 / NEJMc0904283. PubMed PMID:19587352 19. Kocak E, Lute K, Chang X, May KF, Jr, Exten KR, Zhang H, et al. “Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity.” Cancer Res. 2006;66(14):7276-84. Epub 2006 / 07 / 20. doi:10.1158 / 0008-5472. CAN-05-2128. PubMed PMID:16849577 20. Lute KD, May KF, Lu P, Zhang H, Kocak E, Mosinger B, et al. “Human CTLA4-knock-in mice unravel the quantitative link between tumor immunity and autoimmunity induced by anti-CTLA4 antibodies.” Blood. 2005. PubMed PMID: 16037385 21. May KF, Roychowdhury S, Bhatt D, Kocak E, Bai XF, Liu JQ, et al. “Anti-human CTLA4 monoclonal antibody promotes T cell expansion and immunity in a hu-PBL-SCID model: a new method for preclinical screening of costimulatory monoclonal antibodies.” Blood. 2005;105:1114-20. PubMed PMID: 15486062 22. Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J, et al. “High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.” J Biol Chem. 2001;276(9):6591-604. Epub 2000 / 11 / 30. doi:10.1074 / jbc.M009483200. PubMed PMID:11096108 23. Dall'Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA et al. "Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences." J Immunol. 2002;169(9):5171-80. Epub 2002 / 10 / 23. PubMed PMID: 12391234 24. Gao SH, Huang K, Tu H, Adler AS. "Monoclonal antibody humanness score and its applications." BMC biotechnology. 2013;13:55. Epub 2013 / 07 / 06. doi:10.1186 / 1472-6750-13-55. PubMed PMID: 23826749; PubMed Central PMCID: PMC3729710 25. Sun Y, Chen HM, Subudhi SK et al., “Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease,” Nat Med 2002.8:1405-13. 26. Foell J, Strahotin S, O'Neil SP, et al., "CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB×NZW F1 mice." J Clin Invest 2003.111:1505-18. 27. Melero I, Shuford WW, Newby SA, et al., “Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.” Nat Med 1997.3:682-685. 28. May KF, Jr, Chen L, Zheng P, and Liu Y. “Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells.” Cancer Res 2002.62:3459-65 29. Ye Z, Hellstrom I, Hayden-Ledbetter M, et al., “Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB,” Nat Med 2002.8:343-8. 30. Walunas, TL et al., “CTLA4 can function as a negative regulator of T cell activation,” Immunity, 1994.1(5):pp.405-13 31. Krummel MF and J.P. Allison, “CD28 and CTLA4 have opposing effects on the response of T cells to stimulation,” J. Exp. Med., 1995, 182(2):459-65. 32. Anderson, DE et al., “Paradoxical inhibition of T-cell function in response to CTLA4 blockade; heterogeneity within the human T-cell population,” Nat Med, 2000.6(2):pp.211-214. 33. Coyle, AJ et al. (2001) “The Expanding B7 Superfamily: Increasing Complexity In Costimulatory Signals Regulating T Cell Function,” Nature Immunol. 2(3):203-209. 34. Sharpe, AH et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126 35. Collins, M. et al. (2005) “The B7 Family of Immune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7 36. Flajnik, MF et al. (2012) “Evolution of the B7 Family: Co-Evolution of B7H6 and Nkp30, Identification of a New B7 Family Member, B7H7, and of B7's Historical Relationship With the MHC,” Immunogenetics epub doi.org / 10.1007 / s00251-012-0616-2 37. Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation and Anti-Tumor Immunity” Semin. Cancer Biol. 17(4):288-298 38. Flies, DB et al. (2007) “The New B7s: Playing a Pivotal Role in Tumor Immunity,” J. Immunother. 30(3):251-260 39. Ishida, Y. et al. (1992) “Induced Expression of PD-1, a Novel Member of the Immunoglobulin Gene Superfamily, Upon Programmed Cell Death.” EMBO J. 11:3887-3895 40. Agata, Y. et al. (1996) "Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes." Int. Immunol. 8(5):765-772 41. Yamazaki, T. et al. (2002) “Expression of Programmed Death 1 Ligands by Murine T Cells and APCs” J. Immunol. 169:5538-5545 42. Nishimura, H. et al. (2000) “Facilitation of Beta Selection and Modification of Positive Selection in the Thymus of PD-1-Deficient Mice.” J. Exp. Med. 191:891-898 43. Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation and Anti-Tumor Immunity” Semin. Cancer Biol. 17(4):288-298 44. Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). 45. Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917 46. ​​Padlan, 1991, Molecular Immunology 28(4 / 5):489-498 47. Studnicka et al., 1994, Protein Engineering 7:805 48. Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969 49. Keler, T. et al., “Activity and safety of CTLA4 blockade combined with vaccines in cynomolgus macaques,” J. Immunol. 171, 6251–6259 (2003). 50. Wing, K. et al., "CTLA4 control over Foxp3+ regulatory T cell function," Science 322, 271-275, doi:10.1126 / science.1160062 (2008) 51. Schwartz, RS. “The new immunology—the end of immunosuppressive drug therapy?” N. Engl. J. Med. 340, 1754–1756, doi:10.1056 / NEJM199906033402209 (1999) 52. Simpson, TR et al., “Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA4 therapy against melanoma,” J. Exp. Med. 210, 1695–1710, doi:10.1084 / jem.20130579 (2013) 53. Selby, MJ et al., “Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells,” Cancer Immunology Research 1, 32-42, doi:10.1158 / 2326-6066.CIR-13-0013 (2013) 54. Maker, AV, Attia, P., and Rosenberg, SA. “Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA4 blockade.” J. Immunol. 175, 7746-7754 (2005). 55. Korman, AJ, Peggs, KS, and Allison, JP, “Checkpoint blockade in cancer immunotherapy,” Adv. Immunol. 90, 297–339, doi:10.1016 / S0065-2776(06)90008-X (2006). 56. Ribas, A. et al., “Tremelimumab (CP-675, 206), a cytotoxic T lymphocyte-associated antigen 4-blocking monoclonal antibody in clinical development for patients with cancer,” Oncologist 12,873-883,doi:10.1634 / theoncologist.12-7-873(2007) 57. Ribas, A. et al., “Phase III randomized clinical trial comparing tremelimumab with standard-of-care chemotherapy in patients with advanced melanoma,” J. Clin. Oncol. 31, 616–622, doi:10.1200 / JCO.2012.44.6112 (2013) 58. Lee, KM et al., "Molecular basis of T cell inactivation by CTLA4" [In Process Citation] Science 282, 2263-2266 (1998) 59. Marengere, LE et al., "Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA4" [published errata appear in Science 1996 Dec 6;274(5293)1597 and 1997 Apr 4;276(5309):21] Science 272, 1170-1173 (1996). 60. Liu, Y. "Is CTLA4 a negative regulator for T-cell activation?" Immunol. Today 18, 569-572 (1997) 61. Tivol, EA et al., “Loss of CTLA4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA4,” Immunity 3, 541–547 (1995) 62. Waterhouse, P. et al., "Lymphoproliferative disease with early mortality in CTLA4-deficient mice." [See comments] Science 270, 985-988 (1995) 63. Bachmann, M.F., Kohler, G., Ecabert, B., Mak, T.W., and Kopf, M., “Cutting edge: lymphoproliferative disease in the absence of CTLA4 is not T cell autonomous,” J. Immunol. 163, 1128–1131 (1999). 64. Bachmann, MF et al., “Normal pathogen-specific immune responses mounted by CTLA4-deficient T cells: a paradigm reconsidered,” Eur. J. Immunol. 31, 450-458 (2001). 65. Nguyen, TV, Ke, Y., Zhang, EE, and Feng, GS. “Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals.” J. Immunol. 177, 5990-5996 (2006). 66. Qureshi, OS et al., “Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4,” Science 332, 600-603, doi:10.1126 / science.1202947 (2011) 67. Ueda, H. et al., "Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease," Nature 423, 506-511 (2003) 68. Magistrelli, G. et al., "A soluble form of CTLA-4 generated by alternative splicing is expressed by nonstimulated human T cells." Eur. J. Immunol. 29, 3596-3602, doi:10.1002 / (SICI)1521-4141(199911)29:11<3596::AID-IMMU3596>3.0.CO;2-Y(1999) 69. Kremer, JM et al., "Treatment of rheumatoid arthritis by selective inhibition of T-cell activation with fusion protein CTLA4Ig," N. Engl. J. Med. 349, 1907-1915, doi:10.1056 / NEJMoa035075 (2003) 70. Abrams, JR. “CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris.” J. Clin. Invest. 103, 1243–1252, doi:10.1172 / JCI5857 (1999) 71. Gerold, KD et al., “The soluble CTLA-4 splice variant protects from type 1 diabetes and potentiates regulatory T-cell function,” Diabetes 60, 1955–1963, doi:10.2337 / db11-0130 (2011) 72. Peach RJ, Bajorath J, Brady W, Leytze G, Greene J, Naemura J, et al., "Complementarity determining region 1 (CDR1)-and CDR3-analogous regions in CTLA-4 and CD28 determine the binding to B7-1." J Exp Med. 1994;180(6):2049-58. 73. van Elsas, A., Hurwitz, AA, and Allison, JP. “Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.” J. Exp. Med. 190, 355-366 (1999). 74. van Elsas, A. et al., “Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy,” J. Exp. Med. 194, 481-489. (2001) 75. Karandikar, NJ, Vanderlugt, CL, Walunas, TL, Miller, SD, and Bluestone, JA. "CTLA-4: a negative regulator of autoimmune disease." J. Exp. Med. 184, 783-788 (1996). 76. Luhder, F., Chambers, C., Allison, JP, Benoist, C., and Mathis, D., “Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells,” Proc. Natl. Acad. Sci. USA 97, 12204-12209 (2000). 77. Hurwitz, AA, Sullivan, TJ, Sobel, RA, and Allison, JP. “Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB / c mice.” Proc. Natl. Acad. Sci. USA 99, 3013-3017 (2002). 78. Piganelli, JD, Poulin, M., Martin, T., Allison, JP, and Haskins, K. “Cytotoxic T lymphocyte antigen 4 (CD152) regulates self-reactive T cells in BALB / c but not in the autoimmune NOD mouse.” J. Autoimmun. 14, 123-131 (2000) 79. Phan, GQ et al., "Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen-4 blockade in patients with metastatic melanoma," Proc Natl Acad Sci USA 100, 8372-8377 (2003) 80. Eisenthal, A. et al., "Antitumor effects of recombinant interleukin-6 expressed in eukaryotic cells," Cancer Immunol. Immunother. 36, 101-107 (1993) 81. Schroff, RW, Foon, KA, Beatty, SM, Oldham, RK, and Morgan, AC Jr., “Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy,” Cancer Res. 45, 879–885 (1985). 82. Kearney, ER et al., “Antigen-dependent clonal expansion of a trace population of antigen-specific CD4+ T cells in vivo is dependent on CD28 costimulation and inhibited by CTLA-4,” J. Immunol. 155, 1032-1036 (1995) 83. Mittler, RS, Bailey, TS, Klussman, K., Trailsmith, MD, and Hoffmann, MK. “Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy.” J. Exp. Med. 190, 1535-1540 (1999)

[0131] The present disclosure also includes the following aspects. <1> (a) a light chain variable region comprising: (i) a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 21; (ii) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, or 38; and (iii) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (b) a heavy chain variable region comprising: (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; (ii) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, 34, or 35; and (iii) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; an anti-CTLA4 antibody, <2> (1) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33, or (2) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35, or (3) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 38, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35. The anti-CTLA4 antibody of claim 1. <3> the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, 71, or 72; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62, 63, or 64; The anti-CTLA4 antibody of claim 1. <4> the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62. The anti-CTLA4 antibody of claim 3. <5> the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64. The anti-CTLA4 antibody of claim 3. <6> the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64. The anti-CTLA4 antibody of claim 3. <7> 7. The anti-CTLA4 antibody according to claim 1, which is capable of binding to human CTLA4. <8> The anti-CTLA4 antibody of any one of claims 1 to 7, characterized by reduced binding to soluble CTLA4 compared to binding to membrane-bound or immobilized CTLA4. <9> An antigen-binding fragment of the anti-CTLA4 antibody according to any one of claims 1 to 8. <10> 10. An anti-cancer agent comprising the anti-CTLA4 antibody according to any one of claims 1 to 8 or the antigen-binding fragment according to claim 9. <11> The anticancer agent of claim 10, which is administered in combination with an additional agent selected from the group consisting of an anti-PD-1 antibody and an anti-4-1BB antibody. <12> The anti-cancer agent of claim 11, wherein the anti-PD-1 antibody or anti-4-1BB antibody and the anti-CTLA4 antibody are combined in a single molecule as a bispecific antibody. <13> The anticancer agent of claim 10, which induces Treg deletion and local T cell activation in the tumor microenvironment.

Claims

1. A pharmaceutical composition for use in treating lung cancer, melanoma, ovarian cancer, or prostate cancer in a subject in need of treatment, comprising a combination of a first nucleic acid having a first nucleic acid sequence encoding an anti-CTLA4 antibody and a second nucleic acid having a second nucleic acid sequence, the first nucleic acid sequence (a) encodes a light chain variable region comprising: (i) a complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO:21; (ii) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, 37, or 38; and (iii) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23; The second nucleic acid sequence (b) encodes a heavy chain variable region comprising (i) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, (ii) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, 34, or 35, and (iii) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

26.

2. (1) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33, or (2) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35; or (3) CDR2 in the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 38, and CDR2 in the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

35. The pharmaceutical composition of claim 1. Claim 3: The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 70, 71, or 72; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62, 63, or 64; The pharmaceutical composition of claim 1. Claim 4: The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62; The pharmaceutical composition of claim 3.

5. The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 71, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64; The pharmaceutical composition of claim 3.

6. The light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 72, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64; The pharmaceutical composition of claim 3.

7. A pharmaceutical composition described in any one of claims 1 to 6, wherein the anti-CTLA4 antibody is capable of binding to human CTLA4.

8. A pharmaceutical composition described in any one of claims 1 to 7, wherein the anti-CTLA4 antibody is characterized by reduced binding to soluble CTLA4 compared to binding to membrane-bound CTLA4 or immobilized CTLA4.

9. The first nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 16, 18, or 20, which encodes an amino acid sequence comprising a variable region set forth in SEQ ID NO: 70, 71, or 72; and / or The second nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 10, 12, or 14, which encodes an amino acid sequence comprising a variable region set forth in SEQ ID NO: 62, 63, or 64; The pharmaceutical composition according to any one of claims 1 to 8.

10. A pharmaceutical composition described in any one of claims 1 to 9, wherein the second nucleic acid sequence comprises a sequence described in SEQ ID NO: 10 encoding an amino acid sequence including a variable region described in SEQ ID NO: 62, or a sequence described in SEQ ID NO: 14 encoding an amino acid sequence including a variable region described in SEQ ID NO:

64.

11. A pharmaceutical composition described in any one of claims 1 to 10, wherein the first nucleic acid sequence comprises a sequence described in SEQ ID NO: 18 encoding an amino acid sequence including a variable region described in SEQ ID NO: 71, or a sequence described in SEQ ID NO: 20 encoding an amino acid sequence including a variable region described in SEQ ID NO:

72. (1) the first nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 18, which encodes an amino acid sequence comprising a variable region set forth in SEQ ID NO: 71, and the second nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 10, which encodes an amino acid sequence comprising a variable region set forth in SEQ ID NO: 62; or (2) The first nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 18 encoding an amino acid sequence comprising a variable region set forth in SEQ ID NO: 71, and the second nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 14 encoding an amino acid sequence comprising a variable region set forth in SEQ ID NO: 64; or (3) The first nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 20 encoding an amino acid sequence comprising a variable region set forth in SEQ ID NO: 72, and the second nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 14 encoding an amino acid sequence comprising a variable region set forth in SEQ ID NO: 64; A pharmaceutical composition according to any one of claims 1 to 11.

13. A pharmaceutical composition for use in treating lung cancer, melanoma, ovarian cancer, or prostate cancer in a subject in need of treatment, comprising a nucleic acid sequence encoding an antigen-binding fragment of an anti-CTLA4 antibody encoded by the nucleic acid of any one of claims 1 to 12.

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