Anti–CTLA-4 antibody

Abstract

Provided is a novel anti-CTLA-4 antibody.　Specifically provided is an anti-CTLA-4 antibody or an antigen-binding fragment thereof, the anti-CTLA-4 antibody comprising: (a) an H chain which has an H chain variable region that includes CDR-H1 comprising the amino acid sequence NYY, CDR-H2 comprising the amino acid sequence represented by SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence represented by SEQ ID NO: 21; and (b) an L chain which has an L chain variable region that includes CDR-L1 comprising the amino acid sequence SKY, CDR-L2 comprising the amino acid sequence SGS, and CDR-L3 comprising the amino acid sequence represented by SEQ ID NO: 20. Also provided is a pharmaceutical composition which contains the anti-CTLA-4 antibody or an antigen-binding fragment thereof as an active ingredient. Also provided is a method for producing the anti-CTLA-4 antibody or an antigen-binding fragment thereof.

Description

Anti-CTLA-4 antibody

[0001] The present invention relates to an anti-CTLA-4 antibody.

[0002] Malignant tumors (cancer) account for a large proportion of the causes of death in dogs, and this tendency is particularly prominent in elderly individuals. Surgical treatment, chemotherapy, and radiotherapy are performed for canine tumors, but there are also many refractory tumors. In human medicine, in addition to these treatment methods, the application of immunotherapy has advanced. In particular, immune checkpoint inhibitors such as anti-Programmed cell death 1 (PD-1) antibody, anti-PD-ligand 1 (PD-L1) antibody, and anti-Cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody have been put into practical use as therapeutic drugs for many cancer types including malignant melanoma and lung cancer. So far, it has been reported that an anti-PD-L1 antibody drug (canine chimeric anti-PD-L1 antibody) is promising as a therapeutic drug for tumors such as malignant melanoma in dogs (Non-Patent Documents 1, 2, 3). However, since the efficacy rate remains constant, the development of new therapeutic drugs for activating further anti-tumor immunity is required. Anti-CTLA-4 antibody drugs that inhibit the immunosuppressive receptor CTLA-4 have reported good treatment results in combination with anti-PD-1 / PD-L1 antibody drugs and are used as therapeutic drugs for human malignant melanoma and the like. However, no anti-CTLA-4 antibody that can be used for the treatment of malignant tumors in dogs has been reported.

[0003] Maekawa N, Konnai S, Takagi S, Kagawa Y, Okagawa T, Nishimori A, Ikebuchi R, Izumi Y, Deguchi T, Nakajima C, Kato Y, Yamamoto K, Uemura H, Suzuki Y, Murata S, Ohashi K. A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma. Sci Rep. 2017 Aug 21;7(1):8951. doi: 10.1038 / s41598-017-09444-2.Maekawa N, Konnai S, Nishimura M, Kagawa Y, Takagi S, Hosoya K, Ohta H, Kim S, Okagawa T, Izumi Y, Deguchi T, Kato Y, Yamamoto S, Yamamoto K, Toda M, Nakajima C, Suzuki Y, Murata S, Ohashi K. PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oral malignant melanoma. NPJ Precise Oncol. 2021 Feb 12;5(1):1 doi: 10.1038 / s41698-021-00147-6.Maekawa N, Konnai S, Hosoya K, Kim S, Kinoshita R, Deguchi T, Owaki R, Tachibana Y, Yokokawa M, Takeuchi H, Kagawa Y, Takagi S, Ohta H, Kato Y, Yamamoto S, Yamamoto K, Suzuki Y, Okagawa T , Murata S , Ohashi K .Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours. PLoS One. 2023 Oct 4;18(10):e0291727.doi: 10.1371 / journal.pone.0291727.

[0004] The present invention aims to provide a novel anti-CTLA-4 antibody.

[0005] The inventors established an anti-canine CTLA-4 rat monoclonal antibody (clone name: 1C5-E5) and used the amino acid sequence of its complementarity-determining region (CDR) to create a canine anti-canine CTLA-4 monoclonal antibody (ca1C5) that is expected to significantly reduce immunogenicity in dogs while maintaining antigen binding ability. The created canine anti-CTLA-4 antibody ca1C5 has a high binding affinity to canine CTLA-4, inhibits the binding of canine CTLA-4 to its ligand (CD80 / CD86), and further enhances the activation of canine immune cells. When the canine anti-CTLA-4 antibody ca1C5 was administered to dogs with oral malignant melanoma that had relapsed during treatment with an anti-PD-L1 antibody, a reduction in tumor size was observed, demonstrating that the canine anti-CTLA-4 antibody ca1C5 obtained by this invention can be used for the treatment of canine tumors. The inventors also produced a feline anti-CTLA-4 antibody, fe1C5. This feline anti-CTLA-4 antibody exhibited high binding affinity to feline CTLA-4, inhibited the binding of feline CTLA-4 to its ligand (CD80 / CD86), and further enhanced the activation of feline immune cells. This invention was completed based on these findings.

[0006] The gist of the present invention is as follows: (1) An anti-CTLA-4 antibody or antigen-binding fragment thereof comprising (a) an H chain having an H chain variable region comprising CDR-H1 comprising the amino acid sequence of NYY, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and (b) an L chain having an L chain variable region comprising CDR-L1 comprising the amino acid sequence of SKY, CDR-L2 comprising the amino acid sequence of SGS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20. (2) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (1), wherein the H chain variable region and the L chain variable region are derived from rat. (3) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (1) or (2), wherein the H chain and L chain each have a constant H chain region and a constant L chain region of an antibody from an animal other than rat. (4) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (3), wherein the animal other than rat is a dog. (5) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of (1) to (4), wherein the H chain variable region contains the amino acid sequence of SEQ ID NO: 29 and the L chain variable region contains the amino acid sequence of SEQ ID NO: 31. (6) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to (1), which is canine-modified. (7) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to (6), wherein the H chain variable region contains the amino acid sequence of SEQ ID NO: 34 and the L chain variable region contains the amino acid sequence of SEQ ID NO: 35. (8) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to (6), wherein the H chain variable region contains the amino acid sequence of SEQ ID NO: 34 and the L chain variable region contains the amino acid sequence of SEQ ID NO: 50 or 51. (9) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of (1) to (8), wherein the H chain constant region contains the amino acid sequence of SEQ ID NO: 36 or 52 and the L chain constant region contains the amino acid sequence of SEQ ID NO: 37 or 53. (10) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (1), comprising an H chain having a variable H chain region containing the amino acid sequence of SEQ ID NO: 34 and a constant H chain region of SEQ ID NO: 52, and an L chain having a variable L chain region containing the amino acid sequence of SEQ ID NO: 51 and a constant L chain region of SEQ ID NO: 53. (11) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (1), which is felineized.(12) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (11), wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 62 and the L chain variable region comprises the amino acid sequence of SEQ ID NO: 63. (13) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to (11), wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 62 and the L chain variable region comprises the amino acid sequence of SEQ ID NO: 70 or 71. (14) The anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of (11) to (13), comprising an H chain having an H chain variable region comprising the amino acid sequence of SEQ ID NO: 62 and an H chain constant region comprising the amino acid sequence of SEQ ID NO: 64, and an L chain having an L chain variable region comprising the amino acid sequence of SEQ ID NO: 63, 70 or 71 and an L chain constant region comprising the amino acid sequence of SEQ ID NO: 65. (15) An anti-CTLA-4 antibody or antigen-binding fragment thereof according to (11), comprising an H chain having a variable H chain region containing the amino acid sequence of SEQ ID NO: 62 and a constant H chain region containing the amino acid sequence of SEQ ID NO: 64, and an L chain having a variable L chain region containing the amino acid sequence of SEQ ID NO: 71 and a constant L chain region containing the amino acid sequence of SEQ ID NO: 65. (16) A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of (1) to (15). (17) A vector comprising the polynucleotide according to (16). (18) A host cell transformed with the vector according to (17). (19) A method for producing an anti-CTLA-4 antibody or antigen-binding fragment thereof, comprising culturing the host cell according to (18) and collecting the anti-CTLA-4 antibody or antigen-binding fragment thereof from the culture. (20) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (15) as an active ingredient. (21) The pharmaceutical composition according to (20) for the prevention and / or treatment of cancer and / or infectious diseases. (22) The pharmaceutical composition according to (21), wherein the cancer and / or infection is selected from the group consisting of neoplastic diseases, leukemia, viral diseases, prion diseases, bacterial diseases, mycoplasma diseases, rickettsial diseases, chlamydia diseases, fungal diseases and protozoal diseases. (23) The pharmaceutical composition according to any one of (20) to (22), which is administered before, after, or concurrently with the administration of a PD-1 / PD-L1 targeting inhibitor. (24) The pharmaceutical composition according to (23), wherein the PD-1 / PD-L1 targeting inhibitor is an antibody or an antigen-binding fragment thereof.(25) The pharmaceutical composition according to (24), wherein the antibody is an anti-PD-1 antibody or an antigen-binding fragment thereof, or an anti-PD-L1 antibody or an antigen-binding fragment thereof. (26) A combination of (a1) a polynucleotide encoding an H chain having a variable H chain region or an antigen-binding fragment thereof, comprising CDR-H1 comprising the amino acid sequence NYY, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and (b1) a polynucleotide encoding an L chain having a variable L chain region or an antigen-binding fragment thereof, comprising CDR-L1 comprising the amino acid sequence SKY, CDR-L2 comprising the amino acid sequence SGS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20. (27) A combination of (a2) a vector incorporating polynucleotides encoding an H chain having a variable H chain region or an antigen-binding fragment thereof, including CDR-H1 containing the amino acid sequence of NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b2) a vector incorporating polynucleotides encoding an L chain having a variable L chain region or an antigen-binding fragment thereof, including CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10. (28) Host cells transformed by a combination of (a2) a vector incorporating polynucleotides encoding an H chain having a variable H chain region or an antigen-binding fragment thereof, including CDR-H1 containing the amino acid sequence of NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b2) a vector incorporating polynucleotides encoding an L chain having a variable L chain region or an antigen-binding fragment thereof, including CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10. (29) A method for producing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in (28) and collecting an anti-CTLA-4 antibody or an antigen-binding fragment thereof from the culture.

[0007] According to the present invention, a novel anti-CTLA-4 antibody was obtained. This antibody can also be used in animals other than rats. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2025-2777, which is the basis of the priority of this application.

[0008] Reaction rate constant of rat anti-dog CTLA-4 monoclonal antibody against dog CTLA-4. SPR analysis was performed using a 1:1 kinetic binding model to calculate the association rate constant (k a ), dissociation rate constant (k d ), and dissociation constant (K D ). Each value represents the mean ± standard deviation of three independent experiments. Inhibition test of rat anti-dog CTLA-4 monoclonal antibody against CTLA-4 / CD80 and CTLA-4 / CD86 binding. The inhibitory ability of anti-CTLA-4 antibody against (a) CTLA-4 / CD80 binding and (b) CTLA-4 / CD86 binding was examined. Anti-CTLA-4 antibody and biotinylated CTLA-4-Ig mixed at each molar concentration ratio were added to plates immobilized with CD80-Ig or CD86-Ig. A color reaction with enzyme-labeled avidin and substrate was performed to detect the binding of CTLA-4-Ig to CD80-Ig or CD86-Ig. Each point was shown as the ratio (%) obtained by dividing the measured absorbance value by the absorbance value without adding the antibody. Rat IgG 2a (Rat IgG 2a ) and rat IgG 2b (Rat IgG 2b ) were used as negative control antibodies. Error bars indicate standard error. Immunostimulatory effect of rat anti-dog CTLA-4 monoclonal antibody 1C5-E5 in dog PBMC. Dog PBMC (n = 9) were stimulated and cultured for 3 days in the presence of superantigen. The amount of IL-2 production was measured by ELISA when 1C5-E5 was added at a final concentration of 10 μg / mL. Rat IgG 2b (Rat IgG 2b) was used. The gray bars show the median for each group. Wilcoxon signed-rank test was used for statistical testing. Design of the canine anti-CTLA-4 antibody ca1C5-E5. The amino acid sequences of the heavy chain (upper panel) and light chain (lower panel) variable regions of ca1C5-E5 were designed using the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5. Gray highlights indicate each complementarity-determining region. Reaction rate constant of the canine anti-CTLA-4 antibody to canine CTLA-4. SPR analysis was performed using a 1:1 kinetic binding model, and the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D The following was calculated: Preparation of the therapeutic canine anti-CTLA-4 antibody ca1C5. ca1C5 was purified from the culture supernatant of stable-expressing cell clones and subjected to SDS-PAGE and CBB staining. (a) Electrophoresis images under non-reducing conditions and (b) reducing conditions are shown. Black arrowheads indicate heterotetramer bands consisting of two heavy chains and two light chains, and white arrowheads indicate bands of heavy chain or light chain monomers. Reaction rate constant of canine anti-CTLA-4 antibody ca1C5 to canine CTLA-4. SPR analysis was performed using a 1:1 kinetic binding model, and the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D The following values ​​were calculated. Each value represents the mean ± standard deviation of three independently conducted experiments. Inhibition tests of the canine anti-CTLA-4 antibody ca1C5 against CTLA-4 / CD80 and CTLA-4 / CD86 binding. The inhibitory ability of the anti-CTLA-4 antibody on (a) CTLA-4 / CD80 binding and (b) CTLA-4 / CD86 binding was examined. Anti-CTLA-4 antibody and biotinylated CTLA-4-Ig, mixed at various molar concentrations, were added to plates immobilized with CD80-Ig or CD86-Ig. CTLA-4-Ig binding to CD80-Ig or CD86-Ig was detected by a color reaction using enzyme-labeled avidin and a substrate. Each point is shown as the percentage obtained by dividing the obtained absorbance value by the absorbance value without antibody addition. Rat IgG was used as a negative control antibody. 2b(Rat IgG 2b) and canine IgG (Dog IgG) were used. Error bars indicate the standard error. Immunoactivating effect of canine-modified anti-CTLA-4 antibody ca1C5 in canine PBMCs. Canine PBMCs (n = 9) were stimulated and cultured for 3 days in the presence of a superantigen. IFN-γ, IL-2, and TNF-α production were measured by ELISA when ca1C5 was added to a final concentration of 10 μg / mL. Canine IgG (Dog IgG) was used as the negative control antibody. Gray bars indicate the median value for each group. Wilcoxon signed-rank test was used for statistical testing. Induction of antibody-dependent cytotoxic activity by canine-modified anti-CTLA-4 antibody ca1C5. Canine PBMCs (n = 6) stimulated and cultured in the presence of IL-2 were co-cultured with CHO-DG44 cells (target cells) that forcibly expressed canine CTLA-4, and the viability rate was calculated by counting the number of surviving target cells after culture. Ca1C5 was added to a final concentration of 10 μg / mL, and canine IgG (Dog IgG) was used as the negative control antibody. The gray bars represent the median values ​​for each group. Wilcoxon's signed-rank test was used for statistical testing. Immunoactivating effects of canine anti-CTLA-4 antibody Ca1C5 and canine chimeric anti-PD-L1 antibody C4G12 in canine PBMCs. Canine PBMCs (n = 9) were stimulated and cultured for 3 days in the presence of superantigens. IFN-γ, IL-2, and TNF-α production were measured by ELISA when Ca1C5 and C4G12 were added to a final concentration of 10 μg / mL, respectively. Canine IgG (Dog IgG) was used as the negative control antibody. The gray bars represent the median values ​​for each group. Wilcoxon's signed-rank test was used for statistical testing, and Holm's method was used for multiplicity adjustment. Changes in body temperature, heart rate, and respiratory rate before and after administration of canine-modified anti-CTLA-4 antibody ca1C5 as monotherapy. Body temperature, heart rate, and respiratory rate were recorded before administration (0 minutes) and at each time point after the start of administration (15, 30, 90, and 150 minutes). Changes in body temperature, heart rate, and respiratory rate before and after administration of canine-modified anti-CTLA-4 antibody ca1C5 and canine chimeric anti-PD-L1 antibody c4G12 as co-administration.Body temperature, heart rate, and respiratory rate were recorded before administration (0 minutes) and at each time point after the start of administration (30, 60, 90, 105, 120, 180, 240 minutes). Changes in the weight of the test animals and the pharmacokinetics of the canine anti-CTLA-4 antibody ca1C5 and the canine chimeric anti-PD-L1 antibody c4G12. (a) The weight of the test animals was measured at each time point. The dotted line shows an approximate straight line. (b) The serum concentration of each antibody was measured by ELISA. The empty marker indicates a value below the lower limit of quantification. Antitumor effect of canine anti-CTLA-4 antibody ca1C5 in dogs with oral malignant melanoma. Ca1C5 was administered in combination with the canine chimeric anti-PD-L1 antibody c4G12 to a dog (miniature dachshund, 17 years old, neutered male) with recurrent oral malignant melanoma. The maximum diameter of the tumor was recorded in lesion-1 (upper right jaw gingiva) and lesion-2 (left corner of the mouth). Comparison of feline and canine CTLA-4 genes. Alignment of predicted amino acid sequences of feline and canine CTLA-4 is shown. Reaction rate constants of rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 against feline and canine CTLA-4. SPR analysis was performed using a 1:1 kinetic binding model to determine the binding rate constant (k). a ), dissociation rate constant (k d ), and the dissociation constant (K DThe following values ​​were calculated. Each value represents the mean ± standard deviation of three independently performed experiments. Design of feline anti-CTLA-4 antibody fe1C5-E5. The amino acid sequences of the heavy chain (upper panel) and light chain (lower panel) variable regions of fe1C5-E5 were designed using the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5. Gray highlighting indicates each complementarity-determining region. Production of feline anti-CTLA-4 antibodies fe1C5-E5, fe1C5-E5-V1, and fe1C5-E5-V2. Fe1C5-E5, fe1C5-E5-V1, and fe1C5-E5-V2 were expressed using a transient expression system in mammalian cells, and each antibody was purified from the culture supernatant. Each antibody was separated by SDS-PAGE and visualized by CBB staining. (a) Electrophoretic images under non-reducing conditions and (b) reducing conditions are shown. For comparison, rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 was used. The reaction rate constants of each anti-CTLA-4 antibody against feline CTLA-4 are shown. SPR analysis was performed using a 1:1 kinetic binding model to determine the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D The following values ​​were calculated. Each value represents the mean ± standard deviation of three independently performed experiments. Preparation of the therapeutic feline anti-CTLA-4 antibody fe1C5. fe1C5 was purified from the culture supernatant of stable-expressing cell clones, separated by SDS-PAGE, and then stained with CBB. (a) Electrophoresis images under non-reducing conditions and (b) reducing conditions are shown. Black arrowheads indicate bands of heterotetramers consisting of two heavy chains and two light chains, and white arrowheads indicate bands of heavy chains or light chain monomers. Reaction rate constant of feline anti-CTLA-4 antibody fe1C5 to feline CTLA-4. SPR analysis was performed using a 1:1 kinetic binding model, and the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K DThe values ​​were calculated. Each value represents the mean ± standard deviation of three independently conducted experiments. Inhibition tests of feline anti-CTLA-4 antibody fe1C5 against CTLA-4 / CD80 and CTLA-4 / CD86 binding. The inhibitory ability of the anti-CTLA-4 antibody on (a) CTLA-4 / CD80 binding and (b) CTLA-4 / CD86 binding was examined. Anti-CTLA-4 antibody and biotinylated CTLA-4-Ig, mixed at various molar concentrations, were added to plates immobilized with CD80-Ig or CD86-Ig. CTLA-4-Ig binding to CD80-Ig or CD86-Ig was detected by a color reaction using enzyme-labeled avidin and a substrate. Each point is shown as the percentage obtained by dividing the obtained absorbance value by the absorbance value without antibody addition. Cat IgG and rat IgG were used as negative control antibodies. 2b (Rat IgG 2b ) was used. Error bars indicate the standard error. * Tukey's multiple comparison test was used to compare the values ​​of each group when p < 0.05 and the molar concentration ratio of antibody / CTLA-4-Ig was 1. Immunoactivating effect of feline anti-CTLA-4 antibody fe1C5 in feline PBMCs. Feline PBMCs (n = 9) were stimulated and cultured for 3 days in the presence of a superantigen. (a) IL-2, (b) IFN-γ, and (c) TNF-α production were measured by ELISA when fe1C5 was added to a final concentration of 10 μg / mL. Cat IgG was used as the negative control antibody. Gray bars indicate the median value for each group. Wilcoxon signed-rank test was used for statistical testing.

[0009] The present invention will be described in detail below.

[0010] The present invention provides an anti-CTLA-4 antibody or its antigen-binding fragment, comprising (a) an H chain having a variable H chain region comprising CDR-H1 comprising the amino acid sequence NYY, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and (b) an L chain having a variable L chain region comprising CDR-L1 comprising the amino acid sequence SKY, CDR-L2 comprising the amino acid sequence SGS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0011] The inventors have established a rat anti-canine CTLA-4 monoclonal antibody (clone name: 1C5-E5) capable of inhibiting the binding of canine CTLA-4 to its ligands CD80 and CD86. The amino acid sequences of CDRs determined by the IMGT method (Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003; 27:55-77), the KABAT method (Kabat EA, Te Wu T, Perry HM, Gottesman KS, Foeller C. Sequences of proteins of immunological interest. Diane Publ Company, 1992), and the Contact method (MacCallum RM, Martin AC, Thornton JM. Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol. 1996 Oct 11;262(5):732-45) are summarized in the table below.

[0012]

[0013] 1C5-E5 comprises (a) an H chain having a variable H chain region including CDR-H1 containing the amino acid sequence NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b) an L chain having a variable L chain region including CDR-L1 containing the amino acid sequence SKY, CDR-L2 containing the amino acid sequence SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 20.

[0014] In the anti-CTLA-4 antibody of the present invention, one, two, three, four, or five amino acids may be deleted, substituted, or added to the amino acid sequences of CDR-H1 to H3 in (a) and CDR-L1 to L3 in (b).

[0015] The anti-CTLA-4 antibody of the present invention may be a rat antibody and may include 1C5-E5. The anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have rat-derived H chain variable regions and L chain variable regions.

[0016] The anti-CTLA-4 antibody or antigen-binding fragment of the present invention may have a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 31. The amino acid sequence of SEQ ID NO: 29 is the amino acid sequence of the heavy chain variable region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5. The amino acid sequence of SEQ ID NO: 31 is the amino acid sequence of the light chain variable region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5.

[0017] The anti-CTLA-4 antibody of the present invention may be any of the following: polyclonal antibody, monoclonal antibody, rat antibody, chimeric antibody (such as rat-dog chimeric antibody or rat-cat chimeric antibody), single-chain antibody, canine antibody, feline antibody, humanized antibody, fully canine antibody, fully feline antibody, or human antibody. When the anti-CTLA-4 antibody of the present invention is a chimeric antibody, it is often a chimera of an antibody produced by an animal that produces CTLA-4 that cross-reacts with rat anti-dog CTLA-4 antibody 1C5-E5, and a rat antibody. Examples of animals include dogs, cats, cattle, sheep, goats, buffalo, horses, mice, hamsters, guinea pigs, ferrets, rabbits, humans, non-human primates, pigs, chickens, and the like.

[0018] An example of a chimeric antibody is one in which the variable region of a rat anti-canine CTLA-4 antibody 1C5-E5 is linked to the constant region of an antibody from an animal other than rat. Therefore, the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have a constant region of the H chain and a constant region of the L chain of an antibody from an animal other than rat, respectively.

[0019] The antigen-binding fragment of the anti-CTLA-4 antibody of the present invention is an antibody fragment having binding affinity to the antigen (CTLA-4), and examples include Fab, F(ab)'2, ScFv, Diabody, VH, VL, Sc(Fv)2, Bispecific sc(Fv)2, Minibody, scFv-Fc monomer, scFv-Fc dimer, etc.

[0020] By designing amino acid sequences (SEQ ID NOs: 46 and 48) that combine the heavy and light chain variable regions (SEQ ID NOs: 29 and 31) of 1C5-E5 (rat anti-canine CTLA-4 monoclonal antibody) with the constant region sequence of canine IgG-B (IGHG2*02) (SEQ ID NOs: 36) and the constant region sequence of canine immunoglobulin λ chain (SEQ ID NOs: 37), a canine chimeric anti-CTLA-4 antibody (ch1C5-E5) can be produced. The nucleotide sequences of the genes encoding the heavy and light chains of ch1C5-E5 are shown in SEQ ID NOs: 47 and 49, respectively.

[0021] The anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may be canine-modified. The amino acid sequences of the heavy and light chain variable regions of the canine-modified anti-CTLA-4 antibody ca1C5-E5 can be designed by transplanting the complementarity-determining regions (SEQ ID NOs: 1-6) contained in the predicted amino acid sequences of the heavy and light chain variable regions of 1C5-E5 (SEQ ID NOs: 29, SEQ ID NOs: 31) into the framework regions of the canine immunoglobulin heavy chain variable region sequence (SEQ ID NOs: 32) and the canine immunoglobulin κ chain variable region sequence (SEQ ID NOs: 33) (SEQ ID NOs: 34 and 35). The obtained variable region sequences can be combined with the canine IgG-B (IGHG2*02) constant region sequence (SEQ ID NOs: 36) and the canine immunoglobulin λ chain constant region sequence (SEQ ID NOs: 37) to form the heavy and light chain sequences of the canine-modified antibody ca1C5-E5 (SEQ ID NOs: 38 and 40). Therefore, the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 34, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 35.

[0022] As the antibody or its antigen-binding fragment becomes canine, its binding affinity to canine CTLA-4 may decrease. Therefore, amino acid substitutions may be introduced into the heavy chain or light chain variable region to restore the binding affinity to canine CTLA-4. The inventors have created mutant canine anti-CTLA-4 antibodies (ca1C5-E5-V1, ca1C5-E5-V2) in which amino acid substitutions have been introduced into the ca1C5-E5 light chain variable region to restore the decrease in the binding affinity of ca1C5-E5 to canine CTLA-4 that occurs with canine transformation. The amino acid sequences of the light chain variable region of ca1C5-E5-V1 and the light chain variable region of ca1C5-E5-V2 are shown in SEQ ID NOs. 50 and 51, respectively. Accordingly, the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 34 and a light chain variable region containing the amino acid sequence of SEQ ID NO. 50 or 51.

[0023] In the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, the constant region of the H chain preferably has the amino acid sequence of the constant region of an immunoglobulin corresponding to human IgG1. The H chain can be divided into γ chain, μ chain, α chain, δ chain, and ε chain depending on the difference in the constant region, and these differences result in the formation of five classes (isotypes) of immunoglobulins: IgG, IgM, IgA, IgD, and IgE.

[0024] In dogs, the following sequences have been identified as the H chains of IgG: IgG-A (corresponding to human IgG2), IgG-B (corresponding to human IgG1), IgG-C (corresponding to human IgG3), and IgG-D (corresponding to human IgG4). In the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, the constant region of the IgG H chain having ADCC / CDC activity is preferred (IgG1 in humans). One of the mechanisms of action of anti-CTLA-4 antibodies used in humans is to eliminate tumor-infiltrating regulatory T cells that highly express CTLA-4 through antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fcγ receptor. If the constant region of an immunoglobulin corresponding to human IgG1 has not been identified, it is preferable to use an immunoglobulin corresponding to human IgG4 that has been mutated in the relevant region to acquire ADCC / CDC activity.

[0025] In cats, three types of IgG H chain sequences have been reported: IgG1a, IgG1b, and IgG2. Of these, IgG1a and IgG1b possess ADCC / CDC activity, while IgG2 does not. Furthermore, IgG1a is thought to be more frequently present than IgG1b. In the examples described later, in order to produce feline antibodies, the IgG1a constant region sequence, which possesses ADCC / CDC activity, was selected as the constant region sequence of the H chain of the anti-CTLA-4 antibody.

[0026] The light chain (L) of an antibody consists of a Kappa chain and a Lambda chain. In the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, the constant region of the L chain may have the amino acid sequence of either the Kappa chain or the Lambda chain. The relative abundance of the Lambda chain is higher in sheep, cats, dogs, horses, and cattle, while the Kappa chain is higher in mice, rats, humans, and pigs. Therefore, the amino acid sequence of the constant region of the chain with the higher abundance may be selected, or the amino acid sequence of the constant region of the same type of chain as the variable region may be selected. In the examples described later, the constant region sequences of the canine and feline immunoglobulin κ chains were selected as the constant region sequences of the L chain of the anti-CTLA-4 antibody in order to produce canine and feline antibodies.

[0027] The anti-CTLA-4 antibody or antigen-binding fragment of the present invention may have a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 36 or 52, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 37 or 53. SEQ ID NO: 36 is the constant region sequence of canine IgG-B (IGHG2*02), and the amino acid sequence of SEQ ID NO: 52 is the constant region sequence of canine IgG-B (IGHG2*01). SEQ ID NO: 37 is the constant region sequence of canine immunoglobulin lambda chain, and the amino acid sequence of SEQ ID NO: 53 is the constant region sequence of canine immunoglobulin κ chain.

[0028] For use in the treatment of dogs, the anti-CTLA-4 antibody or its antigen-binding fragment may contain a heavy chain having a variable region containing the amino acid sequence of SEQ ID NO: 34 and a constant region of the heavy chain of SEQ ID NO: 52, and a light chain having a variable region containing the amino acid sequence of the light chain of SEQ ID NO: 51 and a constant region of the light chain of SEQ ID NO: 53. The entire heavy and light chain regions of the anti-CTLA-4 antibody for canine treatment may contain the amino acid sequences of SEQ ID NO: 54 and 56, respectively. Sequences of the nucleotide sequences encoding these amino acid sequences, optimized for expression in Chinese hamster-derived cells using codons, are shown in SEQ ID NO: 55 and SEQ ID NO: 57.

[0029] The anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may be felineized. By transplanting the complementarity-determining regions (SEQ ID NOs: 1-6) contained in the predicted amino acid sequences of the heavy and light chain variable regions of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 (SEQ ID NOs: 29, 31) into the framework regions of the feline immunoglobulin heavy chain variable region sequence (SEQ ID NOs: 60) and the feline immunoglobulin κ chain variable region sequence (SEQ ID NOs: 61), the felineized anti-CTLA-4 antibody heavy and light chain variable region amino acid sequences can be designed (SEQ ID NOs: 62 and 63). The obtained variable region sequences can be combined with the feline IgG1a constant region sequence (SEQ ID NOs: 64) and the feline immunoglobulin κ chain constant region sequence (SEQ ID NOs: 65) to form the heavy and light chain sequences (SEQ ID NOs: 66 and 68) of the felineized anti-CTLA-4 antibody 1C5-E5 (fe1C5-E5). Therefore, the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 63.

[0030] As the antibody or its antigen-binding fragment becomes feline, its binding affinity to feline CTLA-4 may decrease. Therefore, amino acid substitutions may be introduced into the heavy chain or light chain variable region to restore the binding affinity to feline CTLA-4. To restore the decreased binding affinity of fe1C5-E5 to canine CTLA-4 due to felineization, the inventors have created mutant feline anti-CTLA-4 antibodies (fe1C5-E5-V1, fe1C5-E5-V2) in which amino acid substitutions have been introduced into the fe1C5-E5 light chain variable region. The amino acid sequences of the light chain variable region of fe1C5-E5-V1 and the light chain variable region of fe1C5-E5-V2 are shown in SEQ ID NOs. 70 and 71, respectively. Accordingly, the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may have a heavy chain variable region containing the amino acid sequence of SEQ ID NO. 62 and a light chain variable region containing the amino acid sequence of SEQ ID NO. 70 or 71.

[0031] The anti-CTLA-4 antibody or antigen-binding fragment of the present invention may include a heavy chain having a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 62 and a constant heavy chain region containing the amino acid sequence of SEQ ID NO: 64, and a light chain having a variable light chain region containing the amino acid sequence of SEQ ID NO: 63, 70, or 71 and a constant light chain region containing the amino acid sequence of SEQ ID NO: 65.

[0032] For use in the treatment of cats, the anti-CTLA-4 antibody or its antigen-binding fragment may contain a heavy chain having a variable region containing the amino acid sequence of SEQ ID NO: 62 and a constant region containing the amino acid sequence of SEQ ID NO: 64, and a light chain having a variable region containing the amino acid sequence of SEQ ID NO: 71 and a constant region containing the amino acid sequence of SEQ ID NO: 65. The entire heavy and light chain regions of the anti-CTLA-4 antibody for cat treatment may contain the amino acid sequences of SEQ ID NO: 66 and 74, respectively. Sequences of the nucleotide sequences encoding these amino acid sequences, optimized for expression in Chinese hamster-derived cells, are shown as SEQ ID NO: 76 and SEQ ID NO: 77.

[0033] The anti-CTLA-4 antibody of the present invention preferably has a quadruple-chain structure consisting of two light chains and two heavy chains.

[0034] In the amino acid sequence that identifies the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, one, two, three, four, five, or more (at most about ten) amino acids may be deleted, substituted, or added. Furthermore, the amino acid sequence that identifies the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention may be replaced with a sequence having 90% or more sequence identity (for example, 90-95%, 95-97%, 97% or more, 98% or more, 99% or more). An anti-CTLA-4 antibody or its antigen-binding fragment identified by a sequence with 90% or more sequence identity is also included in the present invention. The identity of the two sequences can be determined using BLAST.

[0035] In addition to the amino acid sequences of the constant L and H chain regions mentioned above, the amino acid and nucleotide sequences of the constant L and H chain regions of various animals can be obtained from known databases and used.

[0036] The anti-CTLA-4 antibody or its antigen-binding fragment of the present invention can be produced, for example, as follows: Synthesize an artificial gene encoding the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, insert the artificial gene into a vector (e.g., plasmid), introduce it into a host cell (e.g., a mammalian cell such as a CHO cell), culture the host cell, and collect the anti-CTLA-4 antibody or its antigen-binding fragment from the culture.

[0037] The nucleotide sequences of the cDNA encoding the variable regions of the heavy and light chains of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 are shown in SEQ ID NO: 28 and SEQ ID NO: 30, respectively.

[0038] The nucleotide sequences encoding the heavy and light chain amino acid sequences (SEQ ID NOs. 38 and 40) of the canine-derived anti-CTLA-4 antibody ca1C5-E5 are shown as SEQ ID NOs. 39 and 41, respectively, with codons optimized for expression in Chinese hamster-derived cells.

[0039] The nucleotide sequences encoding the light chain amino acid sequences (SEQ ID NO: 42 and SEQ ID NO: 44) of the mutant canine anti-CTLA-4 antibodies (ca1C5-E5-V1 and ca1C5-E5-V2) are shown in SEQ ID NO: 43 and SEQ ID NO: 45, respectively.

[0040] The gene fragments encoding the heavy chain and light chain of the canine chimeric anti-CTLA-4 antibody (ch1C5-E5) are shown in SEQ ID NOs. 47 and 49, respectively.

[0041] The heavy and light chain variable regions of ca1C5-E5-V2 (SEQ ID NOs. 34 and 51) were combined with the constant region sequence of canine IgG-B (IGHG2*01) (SEQ ID NOs. 52) and the constant region sequence of canine immunoglobulin κ chain (SEQ ID NOs. 53) to design the amino acid sequence of the therapeutic canine anti-CTLA-4 antibody ca1C5 (SEQ ID NOs. 54 and 56). Codon-optimized sequences for expression in Chinese hamster-derived cells of the nucleotide sequence encoding this amino acid sequence are shown in SEQ ID NOs. 55 and 57.

[0042] The nucleotide sequences encoding the heavy chain and light chain amino acid sequences (SEQ ID NOs. 66 and 68) of the feline anti-CTLA-4 antibody 1C5-E5 (fe1C5-E5) are shown in SEQ ID NOs. 67 and 69, respectively, which are codon-optimized sequences for expression in Chinese hamster-derived cells.

[0043] Overlap PCR using mutation primers (fe1C5L_OL1_F, fe1C5L_OL1_R, fe1C5L_OL2_F, and fe1C5L_OL2_R) (SEQ ID NOs. 100-103) modified the gene sequences encoding the mutant feline anti-CTLA-4 antibodies (fe1C5-E5-V1 and fe1C5-E5-V2) light chains (SEQ ID NOs. 72 and 74), as shown in SEQ ID NOs. 73 and 75.

[0044] Sequence numbers 76 and 77 show the codon-optimized sequences for expression in Chinese hamster-derived cells, which encode the heavy and light chain amino acids (SEQ ID NOs. 66 and 74) of fe1C5 (fe1C5-E5-V2).

[0045] These nucleotide sequences may be used to produce the anti-CTLA-4 antibody or its antigen-binding fragment according to the present invention. These nucleotide sequences may be replaced with sequences having 90% or more sequence identity (e.g., 90-95%, 95-97%, 97% or more, 98% or more, 99% or more). The identity of the two sequences can be determined using BLAST.

[0046] The present invention also provides polynucleotides encoding the anti-CTLA-4 antibody or its antigen-binding fragment.

[0047] The polynucleotide of the present invention may have restriction enzyme recognition sites, KOZAK sequences, poly(A) addition signal sequences, promoter sequences, intron sequences, etc., added to it.

[0048] The present invention also provides a vector containing the polynucleotide.

[0049] As vectors, plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19, pSH15), bacteriophages such as lambda phage, retroviruses, animal viruses such as vaccinia virus, and insect pathogenic viruses such as baculovirus can be used. In the examples described later, the pDC62c5-U533 vector (Suzuki, Y., Nakagawa, M., Kameda, Y., Konnai, S., Okagawa, T., Maekawa, N., Goto, S., Sajiki, Y., Ohashi, K., Murata, S., Kitahara, Y. and Yamamoto, K. 2020. Novel vector and use thereof. US patent application No. 17 / 054,936.) was used.

[0050] The vector may also contain promoters, enhancers, splicing signals, polyA addition signals, intron sequences, selection markers, SV40 replication origins, and other elements.

[0051] Furthermore, the present invention also provides host cells transformed with the vector. By culturing these host cells and collecting the antibody or its antigen-binding fragment from the culture, an anti-CTLA-4 antibody or its antigen-binding fragment can be produced. Thus, the present invention also provides a method for producing an anti-CTLA-4 antibody or its antigen-binding fragment, which includes culturing the host cells and collecting the anti-CTLA-4 antibody or its antigen-binding fragment from the culture. In the method for producing an anti-CTLA-4 antibody or its antigen-binding fragment of the present invention, a vector incorporating artificial gene DNA (polynucleotide) containing DNA encoding the light chain and DNA encoding the heavy chain may be transfected into host cells, or a vector incorporating DNA encoding the light chain and a vector incorporating DNA encoding the heavy chain may be co-transfected into host cells. The present invention provides a combination of (a1) a polynucleotide encoding an H chain or an antigen-binding fragment thereof having an H chain variable region including CDR-H1 containing the amino acid sequence NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b1) a polynucleotide encoding an L chain or an antigen-binding fragment thereof having an L chain variable region including CDR-L1 containing the amino acid sequence SKY, CDR-L2 containing the amino acid sequence SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 20.

[0052] Furthermore, the present invention also provides a combination of a vector incorporating (a2) a polynucleotide encoding an H chain or an antigen-binding fragment thereof having an H chain variable region, including CDR-H1 containing the amino acid sequence of NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b2) a polynucleotide encoding an L chain or an antigen-binding fragment thereof having an L chain variable region, including CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10.

[0053] Furthermore, the present invention provides host cells transformed by a combination of a vector incorporating polynucleotides encoding an H chain or antigen-binding fragment thereof having an H chain variable region, including (a2) CDR-H1 containing the amino acid sequence NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and (b2) a vector incorporating polynucleotides encoding an L chain or antigen-binding fragment thereof having an L chain variable region, including CDR-L1 containing the amino acid sequence SKY, CDR-L2 containing the amino acid sequence SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 10. The present invention also provides a method for producing an anti-CTLA-4 antibody or its antigen-binding fragment, comprising culturing the above host cells and collecting an anti-CTLA-4 antibody or its antigen-binding fragment from the culture.

[0054] Examples of host cells include bacterial cells (e.g., Escherichia, Bacillus, Bacillus subtilis, etc.), fungal cells (e.g., yeast, Aspergillus, etc.), insect cells (e.g., S2 cells, Sf cells, etc.), animal cells (e.g., CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), and plant cells. Of these, CHO-DG44 cells (CHO-DG44(dhfr- / -)), which are dihydrofolate reductase-deficient cells, are preferred.

[0055] Recombinant vectors can be introduced into a host by methods described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., calcium phosphate method, DEAE-dextran method, transfection method, microinjection method, lipofection method, electrotoporation method, transduction method, scrape loading method, shotgun method, etc.) or by infection.

[0056] The transformants can be cultured in a culture medium, and the anti-CTLA-4 antibody or its antigen-binding fragment of the present invention can be collected from the culture. If the anti-CTLA-4 antibody or its antigen-binding fragment is secreted into the culture medium, the medium can be collected, and the anti-CTLA-4 antibody or its antigen-binding fragment can be separated from the medium and purified. If the anti-CTLA-4 antibody or its antigen-binding fragment is produced within the transformed cells, the cells can be lysed, and the anti-CTLA-4 antibody or its antigen-binding fragment can be separated from the lysate and purified.

[0057] Examples of culture media include, but are not limited to, OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium, FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium, ProCHO 5 medium, and Cellvento CHO-100 medium.

[0058] The pH of the culture medium varies depending on the cells being cultured, but generally, a pH of 6.8 to 7.6 is appropriate, and in most cases, a pH of 7.0 to 7.4 is suitable.

[0059] If the cells to be cultured are CHO cells, the culture of CHO cells can be carried out using methods known to those skilled in the art. For example, they can usually be cultured in an atmosphere with a gas phase CO2 concentration of 0-40%, preferably 2-10%, at 30-39°C, preferably around 37°C.

[0060] The appropriate culture period is usually 1 day to 3 months, preferably 1 day to 3 weeks.

[0061] The separation and purification of anti-CTLA-4 antibodies or their antigen-binding fragments can be carried out by known methods. Known separation and purification methods include methods that utilize differences in solubility, such as salting out and solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing electrophoresis.

[0062] The anti-CTLA-4 antibody or its antigen-binding fragment of the present invention can be used as an antibody drug for animal or human use. Therefore, the present invention provides a pharmaceutical composition containing the above-mentioned anti-CTLA-4 antibody or its antigen-binding fragment as an active ingredient.

[0063] The pharmaceutical compositions of the present invention can be used for the prevention and / or treatment of cancer and / or infectious diseases. Cancers and / or infectious diseases include neoplastic diseases (for example, malignant melanoma, lung cancer, stomach cancer, kidney cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer, prostate cancer, liver cancer, biliary tract cancer, pancreatic cancer, thyroid cancer, adrenal cancer, uterine cancer, testicular cancer, colorectal cancer, head and neck cancer, skin cancer, gastrointestinal stromal tumors, multiple myeloma, bone and soft tissue tumors, brain tumors, eye tumors, malignant mesothelioma, retroperitoneal tumors, malignant melanoma, transitional cell carcinoma, squamous cell carcinoma, anal sac adenocarcinoma, nasal cavity adenocarcinoma, soft tissue sarcoma, osteosarcoma, angiosarcoma, histiocytic sarcoma, mast cell tumor, fibrosarcoma, thymoma, and transplantable genital tumors). Feline infectious diseases (e.g., lymphoma), leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), viral diseases (e.g., rabies, canine distemper, canine parvovirus infection, canine infectious hepatitis, canine infectious laryngotracheitis, canine parainfluenza virus infection, canine herpesvirus infection, canine coronavirus infection, feline leukemia virus infection, feline immunodeficiency virus infection, feline panleukopenia, feline infectious peritonitis / feline enteric coronavirus infection, feline calicivirus disease, feline viral rhinotracheitis, canine viral papilloma) Diseases such as canine respiratory coronavirus infection, feline formy virus infection, feline poxvirus disease, severe fever with thrombocytopenia syndrome, rotavirus disease, Borna disease virus infection, Aujeszky's disease, etc.), prion diseases (e.g., feline spongiform encephalopathy, etc.), bacterial diseases (e.g., canine leptospirosis, canine brucellosis, canine Lyme disease, canine and feline Campylobacter enteritis, canine and feline salmonella infection, canine and feline Bordetella disease, cat scratch disease, canine and feline pasteurellosis, canine and feline atypical mycobacterial infection, tetanus, etc.), mycoplasma diseases (e.g., feline hemoplasmosis) Infections such as canine infections, rickettsial diseases (e.g., canine ehrlichiosis, Rocky Mountain spotted fever, salmon poisoning, etc.), chlamydial diseases (e.g., feline chlamydial disease, etc.), fungal infections (e.g., canine and feline cryptococcosis, canine and feline dermatophytosis, canine and feline histoplasmosis, canine and feline candidiasis, canine and feline Malasseziasis, canine and feline Pneumocystis pneumonia, canine and feline blastomycosis, canine and feline coccidioidomycosis, canine and feline sporotrichosis, canine and feline linosporidiasis, canine and feline protothecasis, aspergillosis, etc.), protozoal diseases (e.g.,Examples of malignant melanomas include canine and feline toxoplasmosis, canine and feline intestinal protozoal infections, canine and feline babesiosis, canine and feline cryptosporidiosis, canine neosporasis, canine and feline intestinal coccidiosis, canine and feline trypanosomiasis, canine leishmaniasis, canine and feline encephalitozoonosis, canine hepatozoonosis, free-living amoebic infection, and cytozoonosis. Cancer may also be accompanied by distant metastases such as lymph node metastasis and lung metastasis. Sites where malignant melanoma can develop include the oral cavity, nasal cavity, hairy skin, mucocutaneous junction, nail bed, foot pads, eyeball, digestive tract, and anal sacs.

[0064] The pharmaceutical composition of the present invention may be administered before, after, or simultaneously with the administration of a PD-1 / PD-L1 targeting inhibitor.

[0065] PD-1 (Programmed cell death-1) is a membrane protein expressed on activated T cells and B cells, while its ligand, PD-L1, is expressed on various cells, including antigen-presenting cells such as monocytes and dendritic cells, and cancer cells. Both PD-1 and PD-L1 act as inhibitors that suppress T cell activation. Certain cancer cells and virus-infected cells express the ligand for PD-1, thereby suppressing T cell activation and evading host immune surveillance.

[0066] Examples of PD-1 / PD-L1 targeting inhibitors include substances that specifically bind to PD-1 or PD-L1, such as proteins, polypeptides, oligopeptides, nucleic acids (including natural and artificial nucleic acids), low-molecular-weight organic compounds, inorganic compounds, cell extracts, and extracts from plants, animals, or soil. These substances may be natural or synthetic. Preferred PD-1 / PD-L1 targeting inhibitors are antibodies or their antigen-binding fragments, more preferably antibodies such as anti-PD-1 antibodies or anti-PD-L1 antibodies, or their antigen-binding fragments. The antibodies only need to have inhibitory activity targeting PD-1 / PD-L1, and may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, or human-type antibodies. Methods for producing these antibodies are well known. The antibodies may be derived from any organism, such as humans, mice, rats, rabbits, goats, guinea pigs, dogs, or cattle. The antigen-binding fragment of the antibody is an antibody fragment that has binding affinity to the antigen (PD-1 or PD-L1), and examples include Fab, F(ab)'2, ScFv, Diabody, VH, VL, Sc(Fv)2, Bispecific sc(Fv)2, Minibody, scFv-Fc monomer, and scFv-Fc dimer. In the examples described later, the rat-dog chimeric anti-PD-L1 antibody c4G12 (WO2018 / 034225) was used as the PD-1 / PD-L1 inhibitor. c4G12 is a chimeric antibody that combines the H chain and L chain variable region genes of a rat anti-bovine PD-L1 monoclonal antibody (4G12) capable of inhibiting the binding of canine PD-1 and PD-L1 with the H chain constant region and Lambda chain constant region genes of canine immunoglobulin (IgG-D), respectively. The amino acid sequences of the H chain CDR1-3 (CDRH1-H3), variable and constant regions, and the entire H chain, as well as the L chain CDR1-3 (CDRL1-L3), variable and constant regions, and the entire L chain of the rat-dog chimeric anti-PD-L1 antibody c4G12, are shown in the table below (analyzed by IMGT method).Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003; 27:55-77). In the present invention, an anti-PD-L1 antibody containing any of the amino acid sequences shown in Tables X1-1 and X1-2 below can be used. In these amino acid sequences, one, two, three, four, five or more (at most about ten) amino acids may be deleted, substituted, or added. Examples of anti-PD-L1 antibodies containing any of the amino acid sequences shown in Tables X1-1 and X1-2 below include anti-PD-L1 antibodies having CDR-H1, H2, H3 and CDR-L1, L2, L3 as shown in Tables X1-1 and X1-2 below, anti-PD-L1 antibodies having a variable H chain region and a variable L chain region, and anti-PD-L1 antibodies having the entire H chain (including the variable H chain region and the constant H chain region) and the entire L chain (including the variable L chain region and the constant L chain region). Anti-PD-L1 antibodies containing any of the amino acid sequences shown in Tables X1-1 and X1-2 below include rat-dog chimeric anti-PD-L1 antibodies, rat-bovine chimeric anti-PD-L1 antibodies, and chimeric anti-PD-L1 antibodies from animals other than rats, dogs, and bovines (e.g., cats, sheep, goats, buffalo, horses, mice, hamsters, guinea pigs, ferrets, rabbits, humans, non-human primates, pigs, chickens, etc.). Anti-PD-L1 antibodies should preferably have a quadruple-chain structure with two light chains and two heavy chains. Anti-PD-L1 antibodies can be manufactured by the methods described in WO2018 / 034225 or Sci Rep 7, 8951 (2017) or by similar methods.

[0067]

[0068]

[0069] Alternatively, rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 (WO2018 / 034226) may be used as a PD-1 / PD-L1 inhibitor. ch5D2 is a chimeric antibody that combines the H chain and L chain variable region genes of a rat anti-bovine PD-1 monoclonal antibody (5D2), which is capable of inhibiting the binding of bovine PD-1 and PD-L1, with the H chain constant region and Lambda chain constant region genes of bovine immunoglobulin (IgG1), respectively. The amino acid sequences of the H chain CDR1-3 (CDRH1-H3), H chain variable and constant regions, the entire H chain, L chain CDR1-3 (CDRL1-L3), L chain variable and constant regions, and the entire L chain of the rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 are shown in the table below (analyzed by IMGT method. Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003; 27:55-77). In the present invention, an anti-PD-1 antibody containing any of the amino acid sequences shown in Table X2 below can be used. In these amino acid sequences, one, two, three, four, five or more (at most about 10) amino acids may be deleted, substituted, or added. Anti-PD-1 antibodies containing any of the amino acid sequences shown in Table X2 below include anti-PD-1 antibodies having CDR-H1, H2, H3 and CDR-L1, L2, L3 as shown in Table X2 below, anti-PD-1 antibodies having a variable H chain region and a variable L chain region, and anti-PD-1 antibodies having the entire H chain region (including the variable H chain region and the constant H chain region) and the entire L chain region (including the variable L chain region and the constant L chain region). Anti-PD-1 antibodies containing any of the amino acid sequences shown in Table X2 below include rat-bovine chimeric anti-PD-1 antibodies as well as chimeric anti-PD-1 antibodies from animals other than rats and bovines (e.g., dogs, cats, etc.). Anti-PD-1 antibodies are preferably quadruple-chained, with two L chains and two H chains.Anti-PD-1 antibodies can be manufactured by the method described in WO2018 / 034226 or by a similar method.

[0070]

[0071] In the pharmaceutical composition of the present invention, a PD-1 / PD-L1 targeting inhibitor and an anti-CTLA-4 antibody or its antigen-binding fragment can be used in combination or as a combination drug.

[0072] When using a PD-1 / PD-L1 targeting inhibitor in combination with an anti-CTLA-4 antibody or its antigen-binding fragment, it is preferable to administer the PD-1 / PD-L1 targeting inhibitor and the anti-CTLA-4 antibody or its antigen-binding fragment separately.

[0073] When combining a PD-1 / PD-L1 targeting inhibitor with an anti-CTLA-4 antibody or its antigen-binding fragment, it is preferable to create a combination drug containing both the PD-1 / PD-L1 targeting inhibitor and the anti-CTLA-4 antibody or its antigen-binding fragment.

[0074] The pharmaceutical composition of the present invention is administered to a subject or test animal systemically or topically, orally or parenterally.

[0075] PD-1 / PD-L1 inhibitors should be dissolved in a buffer such as PBS, physiological saline, or sterile water, filtered and sterilized as needed, and then administered to test animals (including humans) by injection. Additives (e.g., salts, sugars, chelating agents, colorants, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonic agents, pH adjusters, etc.) may also be added to this solution. Administration routes include intravenous, intramuscular, peritoneal, subcutaneous, and intradermal administration, and nasal or oral administration is also permitted.

[0076] The content of PD-1 / PD-L1-targeting inhibitors in formulations varies depending on the type of formulation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. The formulations are preferably prepared as unit-dose formulations.

[0077] The dosage, number of doses, and frequency of administration of PD-1 / PD-L1 targeting inhibitors (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies) vary depending on the symptoms, age, weight, administration method, and form of administration of the test animal or subject. For example, it is generally recommended to administer 0.1 to 100 mg / kg body weight, preferably 1 to 10 mg / kg body weight, per adult animal at least once, at a frequency that yields the desired effect.

[0078] The anti-CTLA-4 antibody or its antigen-binding fragment may be included in a formulation containing a PD-1 / PD-L1 targeting inhibitor, but it is preferable to dissolve it separately in a buffer such as PBS, physiological saline, or sterile water, filter it for sterilization if necessary, and then administer it to test animals (including humans) by injection. Additives (e.g., salts, sugars, chelating agents, colorants, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonic agents, pH adjusters, etc.) may also be added to this solution. Administration routes include intravenous, intramuscular, peritoneal, subcutaneous, and intradermal administration, and it may also be administered intranasally or orally.

[0079] The content of anti-CTLA-4 antibody or its antigen-binding fragment in the formulation varies depending on the type of formulation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. The formulation is preferably prepared into a unit-dose formulation.

[0080] The dosage, number of administrations, and frequency of administration of anti-CTLA-4 antibody or its antigen-binding fragment will vary depending on the symptoms, age, weight, administration method, and form of administration of the test animal or subject. For example, typically, an adult animal or adult should be given at least once at a frequency that allows the desired effect to be confirmed, equivalent to 0.01 to 100 mg / kg body weight of the active ingredient, preferably about 0.1 to 10 mg / kg body weight, more preferably about 1 to 3 mg / kg body weight.

[0081] The appropriate ratio (by mass) of a PD-1 / PD-L1 targeting inhibitor to an anti-CTLA-4 antibody or its antigen-binding fragment is approximately 1:100 to 1000:1, preferably 1:10 to 100:1, and more preferably 1:3 to 20:1.

[0082] The present invention provides a method for the prevention and / or treatment of cancer and / or infectious diseases, comprising administering an anti-CTLA-4 antibody or its antigen-binding fragment in a pharmaceutically effective amount to a subject or test animal at any time before, after, or concurrently with the administration of a PD-1 / PD-L1-targeting inhibitor.

[0083] Furthermore, the present invention provides the use of an anti-CTLA-4 antibody or its antigen-binding fragment for the prevention and / or treatment of cancer and / or infectious diseases, wherein the anti-CTLA-4 antibody or its antigen-binding fragment is administered before, after, or concurrently with the administration of a PD-1 / PD-L1-targeting inhibitor.

[0084] Furthermore, the present invention provides the use of an anti-CTLA-4 antibody or its antigen-binding fragment for use in methods for the prevention and / or treatment of cancer and / or infectious diseases, wherein the anti-CTLA-4 antibody or its antigen-binding fragment is administered before, after, or concurrently with the administration of a PD-1 / PD-L1-targeting inhibitor.

[0085] The immunostimulatory effect of PD-1 / PD-L1-targeted inhibitors is enhanced when used in combination with an anti-CTLA-4 antibody or its antigen-binding fragment. Therefore, the present invention provides an immunostimulatory effect enhancer for PD-1 / PD-L1-targeted inhibitors, comprising an anti-CTLA-4 antibody or its antigen-binding fragment.

[0086] The drug of the present invention can be used as a combination drug or in combination with a PD-1 / PD-L1 targeting inhibitor. The combination and combination formulation of a PD-1 / PD-L1 targeting inhibitor with an anti-CTLA-4 antibody or its antigen-binding fragment is described above. In addition to its use as a pharmaceutical, the drug of the present invention can also be used as an experimental reagent.

[0087] The present invention will be described in detail below based on examples. [Example 1] Canine Anti-CTLA-4 Antibody 1. Introduction Activated T cells express immunosuppressive receptors such as programmed death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4). When their respective ligands bind to these receptors, an inhibitory signal is sent, suppressing T cell activation. These immunosuppressive receptors are known to suppress excessive immune responses while also being involved in immune evasion in tumors. In recent years, biopharmaceuticals targeting immunosuppressive receptors and their ligands have been marketed in human medicine and are used to treat various tumors. In this example, with the aim of establishing a novel treatment method for canine tumors, a rat anti-canine CTLA-4 monoclonal antibody (clone name: 1C5-E5) capable of inhibiting the binding of canine CTLA-4 to its ligands CD80 and CD86 was established, and a canine anti-CTLA-4 antibody (ca1C5) was created using its complementarity determining region (CDR). Next, as an indicator of the biological activity of ca1C5, cytokine production in a culture system of canine peripheral blood mononuclear cells was measured. Furthermore, a treatment trial was conducted in which ca1C5 was administered to dogs with oral malignant melanoma that had relapsed during treatment with anti-PD-L1 antibody, and its antitumor effect was verified.

[0088] 2. Materials and Methods 2.1. Establishment of Rat Anti-Canine CTLA-4 Monoclonal Antibodies Rats (WKY / Izm) were immunized by electroporation using a DNA immunization vector into which the gene sequence encoding the extracellular region of canine CTLA-4 (SEQ ID NO: 22, SEQ ID NO: 23) was inserted. Lymphocytes collected from the spleen and iliac lymph nodes were fused with SP2 myeloma cells by PEG to obtain a large hybridoma pool. Monoclonal antibodies were then established by cloning using the methylcellulose method and limiting dilution method (clone names: 1C5-E5, 2C2-H1, 2D1-A3, 2G2-G8, and 2G5-1G6). The isotype of each monoclonal antibody was determined using the Rat Immunoglobulin Isotyping ELISA Kit (BD Pharmingen).

[0089] 2.2. Examination of the binding affinity of anti-CTLA-4 antibodies to canine CTLA-4 by surface plasmon resonance (SPR) analysis To create an expression plasmid for recombinant protein (canine CTLA-4-His) in which polyhistidine (6 × His) is fused to the C-terminus of the extracellular region of canine CTLA-4, PCR was performed using a pMD20 plasmid (Takara Bio Inc.) in which the coding region of canine CTLA-4 (SEQ ID NO: 22, SEQ ID NO: 23) was inserted, with primers (caCTLA-4-His_F and caCTLA-4-His_R) (SEQ ID NO: 82, SEQ ID NO: 83) having recognition sites for restriction enzymes EcoRV-HF (New England Biolabs) and KpnI-HF (New England Biolabs) added to the 5′ end. The obtained PCR products were treated with each restriction enzyme, purified using the FastGene Gel / PCR Extraction Kit (Nippon Genetics Co., Ltd.), and cloned into a pCXN2.1 vector (Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199.) that had undergone similar restriction enzyme treatment. The resulting expression plasmid was purified using the FastGene Xpress Plasmid PLUS Kit (Nippon Genetics Co., Ltd.).

[0090] Next, 7.5 × 10 7Expi293F cells (Thermo Fisher Scientific) were suspended in 25.5 mL of Expi293 Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 30 μg of expression plasmid was introduced using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) to express the target protein. Canine CTLA-4-His was purified from the culture supernatant using TALON Metal Affinity Resin (Takara Bio). After purification, the buffer was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using Amicon Ultra-15 Ultracel-3 (Merck Millipore). The concentration of purified canine CTLA-4-His was determined by measuring the absorbance at 280 nm using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific).

[0091] To investigate the binding affinity (avidity) of each antibody to canine CTLA-4 using canine CTLA-4-His, SPR analysis was performed using the Biacore X100 system (GE Healthcare). Anti-His antibodies were immobilized on Sensor Chip CM5 (GE Healthcare) using the His Capture Kit (GE Healthcare) via amine coupling. Canine CTLA-4-His was captured on the sensor chip as the ligand, and each antibody, ranging from 20 nM to 2-fold serial dilutions, was used as the analyte. Curve fitting was performed on the obtained sensorgrams using a 1:1 kinetic binding model to determine the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D ) was calculated.

[0092] 2.3. Examination of the inhibitory activity of anti-CTLA-4 antibodies against CTLA-4 / CD80 binding and CTLA-4 / CD86 binding The inhibitory activity of anti-CTLA-4 antibodies against CTLA-4 / CD80 binding and CTLA-4 / CD86 binding was examined using recombinant proteins (CD80-Ig, CD86-Ig, and CTLA-4-Ig) in which the extracellular domain of each factor was expressed as a C-terminal rabbit IgG Fc fusion protein.

[0093] To create recombinant protein expression plasmids in which rabbit IgG-Fc is fused to the C-terminus of the extracellular region of canine CTLA-4, CD80, or CD86, PCR was performed using pMD20 plasmids (Takara Bio Inc.) containing the coding regions of canine CTLA-4 (SEQ ID NO: 22, SEQ ID NO: 23), canine CD80 (SEQ ID NO: 24, SEQ ID NO: 25), or canine CD86 (SEQ ID NO: 26, SEQ ID NO: 27) as templates, with primers (caCTLA-4-Ig_F, caCTLA-4-Ig_R, caCD80-Ig_F, caCD80-Ig_R, caCD86-Ig_F, and caCD86-Ig_R) (SEQ ID NOs: 84-89) having recognition sites for restriction enzymes EcoRV-HF (New England Biolabs) and KpnI-HF (New England Biolabs) added to the 5′ end. The obtained PCR products were treated with each restriction enzyme, purified using the FastGene Gel / PCR Extraction Kit (Genetics Japan), and cloned into a pCXN2.1-Rabbit IgG Fc vector (modified from Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199.) that had undergone similar restriction enzyme treatment. The resulting expression plasmid was purified using NucleoBond Xtra Midi (Takara Bio Inc.).

[0094] 7.5×10 7Expi293F cells (Thermo Fisher Scientific) were suspended in 25.5 mL of Expi293 Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 30 μg of each expression plasmid was introduced using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) to express the target protein. The recombinant proteins (CTLA-4-Ig, CD80-Ig, and CD86-Ig) were purified from the resulting culture supernatant using Ab-Capcher ExTra (ProteNova). After purification, the buffer was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using PD midiTrap G-25 (GE Healthcare). The concentrations of the purified recombinant proteins were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0095] 50 μL each of CD80-Ig and CD86-Ig diluted to 1 μg / mL was dispensed into a 96-well ELISA plate (Thermo Fisher Scientific) and reacted at 37°C for 30 minutes to solidify the phase. After five washes, 200 μL of PBS containing 0.05% Tween20 (Kanto Chemical Co.) and 1% bovine serum albumin (BSA) (Sigma-Aldrich) was added, and the mixture was blocked at 37°C for 30 minutes. Simultaneously, in a separate 96-well plate (Corning), biotinylated CTLA-4-Ig using the Lightning-Link Rapid Biotin Conjugation Kit (Innova Biosciences) was mixed with each antibody at molar concentrations of 0, 0.1, 0.5, 1, 2, 5, and 10 (antibody / CTLA-4-Ig), and reacted at 37°C for 30 minutes. The final concentration of biotinylated CTLA-4-Ig was set to 100 pM. Rat IgG was used as the negative control antibody. 2a (BD Biosciences), rat IgG2b BD Biosciences and canine IgG (Jackson ImmunoResearch) were used at the same concentration. After washing a 96-well ELISA plate five times, 50 μL of a mixture of antibody and biotinylated CTLA-4-Ig was added to the plate and reacted at 37°C for 30 minutes. After five washes, 50 μL of NeutrAvidin Horseradish Peroxidase Conjugate (Thermo Fisher Scientific), diluted to 1 μg / mL, was added to the plate and reacted at 37°C for 30 minutes. After five washes, 50 μL of TMB one component substrate (Bethyl Laboratories) was added and reacted in the dark, then 50 μL of 0.18 M H2SO4 was added to stop the reaction, and the absorption wavelength was measured at 450 nm using a microplate reader MTP-900 (Corona Electric). All washings were performed using PBS with 0.05% Tween20 (Kanto Chemical). PBS was used to dilute CD80-Ig and CD86-Ig, while PBS containing 0.05% Tween20 (Kanto Chemical Co., Ltd.) and 1% BSA (Sigma-Aldrich Co., Ltd.) was used to dilute biotinylated CTLA-4-Ig and the antibody.

[0096] 2.4. Investigation of the activation effect of anti-CTLA-4 antibody on canine peripheral blood mononuclear cells (PBMCs) Canine PBMCs were isolated from heparinized canine peripheral blood by density gradient centrifugation using Percoll (GE Healthcare). The isolated PBMCs were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific), antibiotics (streptomycin 100 μg / mL, penicillin 100 U / mL) (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). A superantigen (Staphylococcal enterotoxin B) (Sigma-Aldrich) was added at a final concentration of 5 μg / mL, and the cells were incubated statically for 3 days at 37°C in the presence of 5% CO2. The production of IFN-γ, L-2, and TNF-α in the culture supernatant after adding each anti-CTLA-4 antibody at a final concentration of 10 μg / mL was measured using Canine IFN-gamma DuoSet ELISA (R&D Systems), Canine IL-2 DuoSet ELISA (R&D Systems), and Canine TNF-alpha DuoSet ELISA (R&D Systems). Rat IgG at the same concentration was used as a negative control. 2b Either BD BioXCell (or canine IgG from Jackson ImmunoResearch) was used. A microplate reader MTP-900 (Corona Electric Co., Ltd.) was used to measure absorbance. To investigate the combined effect with anti-PD-L1 antibodies, canine chimeric anti-PD-L1 antibody c4G12 (WO2018 / 034225) was added at a final concentration of 10 μg / mL. Canine IgG (Jackson ImmunoResearch) at the same concentration was used as a negative control antibody.

[0097] 2.5. Production of Canine Anti-CTLA-4 Antibodies Total RNA was extracted from a hybridoma clone producing the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 using TRIzol reagent (Thermo Fisher Scientific), and the nucleotide sequences of cDNA encoding the variable regions of the heavy and light chains (SEQ ID NOs. 28 and 30) were identified using the 5'-Rapid Amplification of cDNA Ends System Version 2.0 (Thermo Fisher Scientific). From the predicted amino acid sequences of the 1C5-E5 heavy and light chains (SEQ ID NOs. 29 and 31), the complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3, and CDR-L1, CDR-L2, CDR-L3) (SEQ ID NOs. 1-6) were predicted according to previously reported methods. (Kabat EA, Te Wu T, Perry HM, Gottesman KS, Foeller C. Sequences of proteins of immunological interest. Diane Publ Company, 1992; Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003; 27:55-77; MacCallum RM, Martin AC, Thornton JM.) Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol. 1996 Oct 11;262(5):732-45.).

[0098] The complementarity-determining regions contained in the predicted amino acid sequences of the heavy and light chain variable regions of 1C5-E5 were transplanted into the framework regions of the canine immunoglobulin heavy chain variable region sequence (SEQ ID NO: 32) and the canine immunoglobulin κ-chain variable region sequence (SEQ ID NO: 33) to design the heavy and light chain variable region amino acid sequences of the canine anti-CTLA-4 antibody ca1C5-E5 (SEQ ID NO: 34 and SEQ ID NO: 35). The obtained variable region sequences were combined with the canine IgG-B (IGHG2*02) constant region sequence (SEQ ID NO: 36) and the canine immunoglobulin λ-chain constant region sequence (SEQ ID NO: 37) to form the heavy and light chain sequences of ca1C5-E5 (SEQ ID NO: 38 and SEQ ID NO: 40).

[0099] The nucleotide sequences encoding these amino acids were optimized for expression in Chinese hamster-derived cells (SEQ ID NOs. 39 and 41), and artificial genes were synthesized (Eurofins Genomics). Each sequence was inserted into a pCAGEN vector (Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci US A. 2004 Jan 6. 101(1):16-22.), and the resulting expression plasmid was purified using NucleoBond Xtra Midi (Takara Bio). Mutant canine anti-CTLA-4 antibody (ca1C5-E5-V1 and ca1C5-E5-V2) light chain (SEQ ID NOs. 42 and 44) ​​expression plasmids were prepared by modifying the gene sequences encoding the light chain variable region (SEQ ID NOs. 43 and 45) using overlap PCR with mutation primers (ca1C5L_OL1_F, ca1C5L_OL1_R, ca1C5L_OL2_F, ca1C5L_OL2_R, ca1C5L_OL3_F, and ca1C5L_OL3_R) (SEQ ID NOs. 90-95), and then inserting the target gene fragments into the pCAGEN vector. 1.5 × 10⁻⁶ 8ExpiCHO-S cells (Thermo Fisher Scientific) were suspended in 25 mL of ExpiCHO Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 10 μg each of antibody heavy chain and light chain expression plasmids were transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) to express the target antibody. From the resulting culture supernatant, ca1C5-E5, ca1C5-E5-V1, and ca1C5-E5-V2 were purified using Ab-Capcher ExTra (ProteNova). After purification, the solvent was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using a PD-10 Desalting Column (GE Healthcare). The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific).

[0100] Furthermore, amino acid sequences were designed (sequences 46 and 48) by combining the heavy and light chain variable regions of 1C5-E5 (sequences 29 and 31) with the canine IgG-B (IGHG2*02) constant region sequence (sequence 36) and the canine immunoglobulin λ chain constant region sequence (sequence 37), and a canine chimeric anti-CTLA-4 antibody (ch1C5-E5) was produced. Gene fragments encoding the heavy and light chains of ch1C5-E5 (sequences 47 and 49) were inserted into a pCAGEN vector, and after expression in ExpiCHO-S cells, the antibody was purified from the culture supernatant.

[0101] 2.6. Establishment of stable expression cells of the therapeutic canine anti-CTLA-4 antibody ca1C5 The heavy and light chain variable regions of ca1C5-E5-V2 (SEQ ID NOs. 34 and 51) were combined with the constant region sequence of canine IgG-B (IGHG2*01) (SEQ ID NOs. 52) and the constant region sequence of canine immunoglobulin κ chain (SEQ ID NOs. 53) to design the amino acid sequence of the therapeutic canine anti-CTLA-4 antibody ca1C5 (SEQ ID NOs. 54 and 56). The nucleotide sequence encoding this amino acid sequence was codon-optimized for expression in Chinese hamster-derived cells (SEQ ID NOs. 55 and 57), and the restriction enzyme recognition sequences of NotI and SbfI (heavy chain) or AscI and AsiSI (light chain) were added to synthesize the artificial gene (Genscript). pDC62c5-U533 vector (Suzuki, Y., Nakagawa, M., Kameda, Y., Konnai, S., Okagawa, T., Maekawa, N., Goto, S., Sajiki, Y., Ohashi, K., Murata, S., Kitahara, Y. and Yamamoto, K. 2020. Novel vector and use thereof. US patent application No. 17 / 054,936.) with restriction enzymes AscI, AsiSI, NotI-HF and SbfI-HF (New England Biolabs) and DNA Ligation Kit<Mighty Mix> The target gene fragment was inserted using Takara Bio (Takara Bio), and the plasmid was purified using NucleoBond Xtra Midi (Takara Bio). The expression plasmid was introduced into CHO-DG44 cells (Thermo Fisher Scientific) using Lipofectamine LTX reagent (Thermo Fisher Scientific), and stable expression clones were established by limiting dilution after changing the medium to CD OptiCHO medium (Thermo Fisher Scientific) containing 4 mM GlutaMAX-I (Thermo Fisher Scientific).The established stable expression clones were incubated in Dynamis medium (Thermo Fisher Scientific) containing 4 mM GlutaMAX-I (Thermo Fisher Scientific) for 0.5 × 10⁻¹⁶ times. 6 The antibodies were suspended at a concentration of cells / mL and cultured with shaking (125 rpm) at 37°C in the presence of 8% CO2 for 14 days. 4 g / L, 4 g / L, and 6 g / L glucose (Fujifilm Wako Pure Chemical Industries) were added on days 3, 5, and 7, respectively, and 3.3% v / v EfficientFeed B+ 3× Supplement (Thermo Fisher Scientific) was added on days 3, 5, 7, and 10, respectively. The culture supernatant obtained after 14 days of shaking culture was subjected to a three-step antibody purification process using protein A affinity chromatography, anion exchange chromatography, and cation exchange chromatography. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific). To confirm the expression and purification of ca1C5, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. An equal volume of 2 × Laemmli Sample Buffer (BIO-RAD) containing 2-Mercaptoethanol (2-ME) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the antibody-containing solution and treated at 96°C for 5 minutes (reducing conditions). A sample solution without 2-ME was also prepared (non-reducing conditions). Subsequently, the sample proteins were separated by electrophoresis using SuperSep Ace 5-20% (Fujifilm Wako Pure Chemical Industries, Ltd.). Precision Plus Protein Standards all blue (Bio-Rad) was used as a molecular weight marker. After electrophoresis, the gels were stained with the Quick-CBB staining kit (Fujifilm Wako Pure Chemical Industries, Ltd.) and then destained in distilled water.

[0102] 2.7. Evaluation of Antibody-Dependent Cytotoxicity Activity by Canine Anti-CTLA-4 Antibody ca1C5 To produce an expression plasmid that expresses canine CTLA-4 on the cell membrane, PCR was performed using a pMD20 plasmid (Takara Bio Inc.) with the coding region of canine CTLA-4 (SEQ ID NO: 22, SEQ ID NO: 23) inserted, with primers (caCTLA-4-EGFP_F and caCTLA-4-EGFP_R) (SEQ ID NO: 96 and SEQ ID NO: 97) that had the restriction enzyme BglII (New England Biolabs) and EcoRI (New England Biolabs) recognition sites added to the 5′ end. After processing the obtained PCR products with each restriction enzyme, they were purified using the FastGene Gel / PCR Extraction Kit (Nippon Genetics Co., Ltd.) and cloned into a pEGFP-N2 vector (Takara Bio Inc.) that had undergone similar restriction enzyme treatment. The obtained expression plasmid was purified using NucleoBond Xtra Midi (Takara Bio Inc.). The expression plasmid was introduced into CHO-DG44 cells (Thermo Fisher Scientific) using Lipofectamine LTX reagent (Thermo Fisher Scientific). Stable expression cells were selected using CD-DG44 medium (Thermo Fisher Scientific) containing G418 (Enzo Life Science) 800 μg / mL, GlutaMAX-I supplement (Thermo Fisher Scientific) 20 mL / L, and 10% Pluronic F-68 (Thermo Fisher Scientific) 18 mL / L, and stable expression cell clones were established by limiting dilution (canine CTLA-4 expressing cells).

[0103] Canine PBMCs isolated according to the method described above were stimulated and cultured for 24 hours in a medium supplemented with recombinant canine IL-2 (Kingfisher Biotech) 200 ng / mL, and non-adherent cells were collected and used as effector cells. Canine CTLA-4 expressing cells (1 × 10⁶) 6Cells (5 × 10) were prepared as target cells, and effector cells (5 × 10) were used. 6 Cells were mixed with 100 μL of the target cell mixture (cells / mL) and cultured for 24 hours (effector:target cell ratio = 5:1). Canine IgG (Jackson ImmunoResearch) was added at a concentration of 10 μg / mL as a negative control antibody for ca1C5. After culturing, cells were stained with Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) and PerCP / Cy5.5-labeled anti-CD14 antibody (Washington State University Monoclonal Antibody Center). The anti-CD14 antibody was labeled with PerCP / Cy5.5 using the Lightning-Link Conjugation Kit (Innova Biosciences). After washing, the target cells were suspended in PBS containing 1% BSA (Sigma-Aldrich) and CountBright Absolute Counting Beads (Thermo Fisher Scientific). The absolute number of viable target cells was calculated by flow cytometry analysis using FACSVerse (BD Biosciences). Cell viability (%) was calculated using the following formula.

[0104] Cell viability (%) = 100 × {(Number of surviving target cells under each condition) / (Number of surviving target cells by PBS)}

[0105] 2.8. Evaluation of the safety and pharmacokinetics of the canine anti-CTLA-4 antibody ca1C5. To evaluate the safety and pharmacokinetics of ca1C5 monotherapy, experimental dogs (beagles, male, 2 years old) were given ca1C5 (1 mg / kg) diluted in physiological saline and administered intravenously over 30 minutes using a syringe pump. Four repeated administrations were performed at 2-week intervals, and serum samples were collected regularly at each time point after administration. Physical examination findings such as body weight, temperature, heart rate, and respiratory rate were recorded, and adverse events were recorded through blood tests, urine tests, chest and abdominal X-rays, and ultrasound examinations. Subsequently, the study proceeded to a combination therapy study with the canine chimeric anti-PD-L1 antibody c4G12. In the combination therapy, c4G12 (5 mg / kg) was administered intravenously over 1 hour, followed by the intravenous administration of ca1C5 (1 mg / kg) over 30 minutes. Five repeated administrations were performed at approximately two-week intervals, and similar sample collection and adverse event recording were carried out. The classification of adverse events by type and grade followed the Veterinary Cooperative Oncology Group-common terminology criteria for adverse events (VCOG-CTCAE) v1.1 (Veterinary Cooperative Oncology Group (VCOG). Veterinary cooperative oncology group-common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.1. Vet Comp Oncol. 2016; 14: 417-446).

[0106] The serum concentrations of the administered antibodies were measured by ELISA. Canine CTLA-4-Ig 1 μg / mL or canine PD-L1-Ig (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, et al. (2014) Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-c Production from Tumor-Infiltrating Cells by PD-L1 Blockade. PLoS ONE 9(6): e98415.) 10 μg / mL were immobilized on a 96-well ELISA plate (Thermo Fisher Scientific) and blocked using a Superblock T20 (Thermo Fisher Scientific). After reacting diluted serum, the reaction was chromogenically developed using HRP-conjugated sheep anti-dog IgG2 antibody (Bethyl Laboratories) or HRP-conjugated goat anti-dog IgG1 antibody (Bethyl Laboratories) and TMB one component substrate (Bethyl Laboratories). The reaction was stopped by adding 0.18 M H2SO4. Measurements were performed at an absorption wavelength of 450 nm using a microplate reader MTP-900 (Corona Electric). All washes were performed using PBS with 0.05% Tween20 (Kanto Chemical).

[0107] 2.9. Evaluation of the efficacy of canine anti-CTLA-4 antibody ca1C5 in dogs with oral malignant melanoma. At the Hokkaido University Veterinary Medical Center, dogs (miniature dachshunds, 17 years old, neutered male) with oral malignant melanoma that relapsed after radiotherapy and during immunotherapy with c4G12 were administered canine chimeric anti-PD-L1 antibody c4G12 (5 mg / kg) and ca1C5 (1 mg / kg) using the method described above. The antibody drugs were administered at 2-week intervals, and the maximum diameter of the tumor was measured using calipers. The tumor reduction effect was assessed according to the Response evaluation criteria for solid tumors in dogs (cRECIST) v1.0 (Nguyen SM, Thamm DH, Vail DM, London CA. Response evaluation criteria for solid tumors in dogs (v1.0): a Veterinary Cooperative Oncology Group (VCOG) consensus document. Vet Comp Oncol. 2015; 13: 176-183), and a partial response was defined as a reduction of 30% or more in the sum of the maximum diameters of each tumor compared to baseline.

[0108] 2.10. Statistical Analysis The Wilcoxon signed-rank test was used for statistical testing. Holm's method was used to adjust for multiples, and a significance level (p) of less than 0.05 was considered statistically significant.

[0109] 3. Results and Discussion 3.1. Establishment of Rat Anti-Canine CTLA-4 Monoclonal Antibodies and Selection Lymphocytes were collected from rats immunized using DNA immunoassay with the extracellular region of canine CTLA-4 as the immunosource, and numerous hybridomas were established by fusing these lymphocytes with mouse myeloma cells. The hybridomas were cloned, and monoclonal antibodies were obtained. To examine the binding affinity of the five established monoclonal antibodies (1C5-E5, 2C2-H1, 2D1-A3, 2G2-G8, and 2G5-1G6) to canine CTLA-4, the reaction rate constants were calculated by SPR analysis using a 1:1 kinetic binding model. Binding rate constants (k) of each antibody a ), dissociation rate constant (k d ), and the dissociation constant (K D The results are shown in Figure 1 (Figure 1). Each monoclonal antibody had a low dissociation constant and was found to bind to canine CTLA-4 with high affinity.

[0110] Next, the inhibitory activity of each monoclonal antibody against CTLA-4 / CD80 binding or CTLA-4 / CD86 binding was investigated. Solutions of biotinylated canine CTLA-4 recombinant protein (CTLA-4-Ig) and each anti-CTLA-4 antibody, pre-reacted at various molar concentrations, were added to ELISA plates immobilized with canine CD80 recombinant protein (CD80-Ig) or canine CD86 recombinant protein (CD86-Ig). Canine CTLA-4-Ig bound to CD80-Ig or CD86-Ig was detected by a color reaction using enzyme-labeled avidin. Each monoclonal antibody exhibited inhibitory activity against both CTLA-4 / CD80 binding and CTLA-4 / CD86 binding, with the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 exhibiting the highest inhibitory effect (Figure 2).

[0111] Furthermore, as an indicator of the immune-activating effect of 1C5-E5, the amount of IL-2 produced in a stimulated culture system of canine PBMCs was compared. In PBMCs cultured with 1C5-E5 added, the IL-2 concentration in the culture supernatant was significantly higher compared to when a negative control antibody was added, revealing that 1C5-E5 has an effect of enhancing the activation of canine immune cells (Figure 3).

[0112] Based on the above findings, 1C5-E5 exhibits high binding affinity to canine CTLA-4 and a strong inhibitory effect on ligand binding to CTLA-4, and enhances the activation of canine immune cells, suggesting its potential use as a therapeutic antibody against canine tumors.

[0113] 3.2. Production of Canine Anti-CTLA-4 Antibody To create an anti-CTLA-4 antibody with reduced immunogenicity in dogs and suitable for repeated administration to dogs, we designed a canine anti-CTLA-4 antibody, ca1C5-E5, by transplanting the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 into a canine antibody sequence (Figure 4). An expression vector for ca1C5-E5 was constructed, transient expression was performed in mammalian cells, and ca1C5-E5 was purified from the culture supernatant. To investigate the binding affinity of ca1C5-E5 to canine CTLA-4, the reaction rate constants for binding of 1C5-E5, canine chimeric 1C5-E5 (ch1C5-E5), and ca1C5-E5 to canine CTLA-4 were calculated using SPR analysis with a 1:1 kinetic binding model. The calculated dissociation constant (K) D ) showed 2.48 × 10⁶ values ​​for rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 and canine chimeric anti-CTLA-4 antibody ch1C5-E5, respectively. -10 M and 1.70 × 10 -10 It was M (Figure 5). On the other hand, the dissociation constant of ca1C5-E5 (K D ) is 8.96 × 10 -10 The antibody was M, which was approximately four times higher than the original 1C5-E5 antibody, suggesting that the binding affinity to canine CTLA-4 decreased in ca1C5-E5 as the antibody became more canine.

[0114] 3.3. Production of Mutant Canine Anti-CTLA-4 Antibodies (ca1C5-E5-V1 and ca1C5-E5-V2) To restore the decreased binding affinity of ca1C5-E5 to canine CTLA-4 due to canine mutation, mutant canine anti-CTLA-4 antibodies (ca1C5-E5-V1 and ca1C5-E5-V2) were produced by introducing amino acid substitutions into the ca1C5-E5 light chain variable region. The amino acid sequences of the light chain variable region of ca1C5-E5-V1 and ca1C5-E5-V2 are shown in SEQ ID NOs. 50 and 51, respectively. To investigate the binding affinity of ca1C5-E5-V1 and ca1C5-E5-V2 to canine CTLA-4, the reaction rate constant was calculated using SPR analysis with a 1:1 kinetic binding model. The calculated dissociation constant (K D ) is 6.22 × 10 for ca1C5-E5-V1 and ca1C5-E5-V2, respectively. -10 M and 2.27 × 10 -10 The result was M (Figure 5). Compared to ca1C5-E5, the dissociation constant of ca1C5-E5-V2 was approximately four times lower, suggesting that its binding affinity to canine CTLA-4 recovered to roughly the same level as that of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5.

[0115] 3.4. Establishment of stable expression cells of the therapeutic canine anti-CTLA-4 antibody ca1C5. Based on the above results, among the canine anti-CTLA-4 antibodies produced (ca1C5-E5, ca1C5-E5-V1, and ca1C5-E5-V2), the variable region of ca1C5-E5-V2 was considered to be the most suitable as the variable region for the therapeutic antibody. A large quantity of the antibody is needed to investigate safety through administration studies using healthy dogs and to investigate therapeutic efficacy through clinical trials in dogs with tumors. Therefore, using the variable region sequence, the amino acid sequences of the heavy chain (SEQ ID NO: 54) and light chain (SEQ ID NO: 56) of the canine anti-CTLA-4 antibody ca1C5 for canine tumor treatment were designed, and stable expression cell clones that highly express ca1C5 were established using the pDC62c5-U533 / CHO-DG44 expression system (Suzuki, Y., Nakagawa, M., Kameda, Y., Konnai, S., Okagawa, T., Maekawa, N., Goto, S., Sajiki, Y., Ohashi, K., Murata, S., Kitahara, Y. and Yamamoto, K. 2020. Novel vector and use thereof. US patent application No. 17 / 054,936.). After 14 days of production culture, ca1C5 was purified from the culture supernatant, and the purified ca1C5 was analyzed by SDS-PAGE. Under non-reducing conditions, bands thought to be from a heterotetramer were observed, while under reducing conditions, bands thought to be from the heavy chain and light chain were observed, respectively (Figure 6). Since the positions of the observed bands were roughly in agreement with the molecular weight calculated from the predicted amino acid sequence, ca1C5 was subjected to the following functional tests.

[0116] 3.5. Examination of the binding affinity of the canine anti-CTLA-4 antibody ca1C5 to canine CTLA-4. To examine the binding affinity of ca1C5 to canine CTLA-4, the reaction rate constant was calculated using SPR analysis with a 1:1 kinetic binding model. The calculated dissociation constant of ca1C5 to canine CTLA-4 (K D ) is 2.80 × 10 -10The dissociation constant of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 is M, and the dissociation constant of 1C5-E5 is 2.63 × 10⁻⁶. -10 It was equivalent to M (Figure 7).

[0117] 3.6. Investigation of the inhibitory activity of the canine anti-CTLA-4 antibody ca1C5 on CTLA-4 / CD80 binding and CTLA-4 / CD86 binding Next, the inhibitory activity of ca1C5 on CTLA-4 / CD80 binding or CTLA-4 / CD86 binding was investigated. A solution of biotinylated canine CTLA-4-Ig and ca1C5 pre-reacted at various molar concentrations was added to an ELISA plate immobilized with canine CD80-Ig or canine CD86-Ig. Canine CTLA-4-Ig bound to CD80-Ig or CD86-Ig was detected by a color reaction using enzyme-labeled avidin. ca1C5 inhibited both CTLA-4 / CD80 binding and CTLA-4 / CD86 binding, and its inhibitory effect was comparable to that of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 (Figure 8).

[0118] 3.7. Examination of the effect of canine anti-CTLA-4 antibody ca1C5 on canine PBMC activation To examine the immune-activating effect of ca1C5, canine PBMCs were cultured for 3 days in the presence of a superantigen, and cytokine concentrations in the culture supernatant were quantified by ELISA. Compared with the addition of canine IgG, which was used as a negative control, the production of IFN-γ, IL-2, and TNF-α increased significantly when ca1C5 was added (Figure 9), suggesting that ca1C5 has the effect of enhancing the activation of canine immune cells.

[0119] 3.8. Investigation of the ability of the canine anti-CTLA-4 antibody ca1C5 to induce antibody-dependent cytotoxic activity in CTLA-4-expressing cells. Anti-CTLA-4 antibodies have been reported to have the effect of eliminating tumor-infiltrating regulatory T cells that highly express CTLA-4 through antibody-dependent cytotoxic activity mediated by binding to the Fcγ receptor. To investigate whether ca1C5 has antibody-dependent cytotoxic activity in CTLA-4-expressing cells, CHO-DG44 cells (target cells) overexpressing canine CTLA-4 and canine PBMCs (effector cells) stimulated with IL-2 were co-cultured, and then the viability of the target cells was calculated to measure cytotoxic activity. When ca1C5 was added to the culture, the viability of the target cells was significantly reduced (Figure 10), indicating that ca1C5 has the ability to induce antibody-dependent cytotoxic activity.

[0120] 3.9. Investigation of the activation effect of canine PBMCs by combining canine anti-CTLA-4 antibody ca1C5 and canine chimeric anti-PD-L1 antibody c4G12. As an indicator of the immune activation effect by combining with an anti-PD-L1 antibody, cytokine concentrations in the culture supernatant were quantified by ELISA when ca1C5 and c4G12 were simultaneously added to the canine PBMC culture system described above. Production of IL-2 and TNF-α was significantly increased by treatment with ca1C5 or c4G12 alone, and a further increase was observed in the combined treatment compared to each treatment alone. Production of IFN-γ was significantly increased in the combined treatment compared to treatment with a negative control antibody (Figure 11). From the above, it was suggested that ca1C5 and c4G12 have the effect of enhancing the activation of canine immune cells even when treated alone, but this effect is further enhanced by combined treatment.

[0121] 3.10. Evaluation of the safety and pharmacokinetics of the canine anti-CTLA-4 antibody ca1C5. One experimental dog (beagle) was repeatedly administered ca1C5 at a dose of 1 mg / kg at 2-week intervals for four doses. No significant changes were observed in body temperature, heart rate, or respiratory rate before and after administration, and no acute adverse events were observed (Figure 12). Blood tests 28 days after the fourth dose showed elevated levels of CRP, an inflammatory marker, but no abnormalities were found in other clinical tests. When the dog was observed without treatment, CRP decreased to within the normal range after 7 days.

[0122] Since a certain level of safety was confirmed with monotherapy, the treatment was transitioned to combination therapy with the canine chimeric anti-PD-L1 antibody c4G12. After four repeated administrations of c4G12 5 mg / kg and ca1C5 1 mg / kg at two-week intervals, no significant changes in body temperature, heart rate, or respiratory rate were observed before and after administration, and no acute adverse events were observed (Figure 13). Fourteen days after the fourth combination administration, a blood test showed elevated CRP levels again, and a chest X-ray on the same day revealed imaging findings of pneumonia. There were no clinical symptoms, only imaging findings, and it was diagnosed as mild pneumonia (pneumonia, grade 1). When the patient was observed without treatment, CRP levels decreased to within the normal range three days later, and the imaging findings on X-ray had disappeared. Therefore, a fifth combination administration was performed, but the increase in CRP levels and the X-ray findings of pneumonia were not reproduced, and no adverse events were observed during the subsequent observation period. Furthermore, the body weight of the test animals increased gradually throughout both the monotherapy and combination therapy periods (Figure 14), suggesting that no serious side effects occurred.

[0123] Serum concentrations of ca1C5 reached their peak immediately after administration and then gradually decreased (Figure 14). Similar pharmacokinetics were observed after the 2nd to 4th doses in repeated administrations at 2-week intervals, and no increase in blood concentration due to accumulation was observed. Similar pharmacokinetics were observed in co-administration with c4G12 (Figure 14), and no interference between antibody drugs was observed.

[0124] Based on the above, ca1C5 was found to be tolerable in dogs when administered alone and in combination with c4G12. Mild pneumonia, thought to be associated with immune activation, was observed in combination administration, but it was within the expected range of type and severity of side effects. Serum antibody concentrations showed the expected pharmacokinetics, and the dose and administration interval were considered appropriate.

[0125] 3.11. Investigation of the antitumor effect of the canine anti-CTLA-4 antibody ca1C5 A 17-year-old neutered male miniature dachshund suffering from oral malignant melanoma (stage II) received radiotherapy at the Hokkaido University Veterinary Medical Center, followed by immunotherapy with the canine chimeric anti-PD-L1 antibody c4G12 (5 mg / kg). Eighteen weeks after the start of c4G12 treatment, recurrence of malignant melanoma was observed in the oral cavity, so ca1C5 (1 mg / kg) was started in combination with c4G12. The lesion in the upper right jaw gingiva (lesion-1), which had a maximum diameter of 18 mm at the start of combination therapy (baseline), had a maximum diameter of 17 mm at 15 weeks of combination therapy, showing a slight reduction with ca1C5 administration. Furthermore, the lesion at the left corner of the mouth (lesion-2), which had a maximum diameter of 12 mm at baseline, completely disappeared at 10 weeks of combination therapy and remained gone at 15 weeks. The sum of the maximum diameters decreased from 30 mm at baseline to 17 mm at 15 weeks of combination therapy (a 43% reduction), and the best overall response was judged to be a partial response (Figure 15). These results demonstrate that ca1C5 exhibits antitumor effects against malignant melanoma in dogs.

[0126] 4. Conclusion The canine anti-CTLA-4 antibody (ca1C5), created using the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5, exhibited binding affinity to canine CTLA-4 and inhibitory effects on canine CTLA-4 / CD80 and CTLA-4 / CD86 binding, comparable to the original rat monoclonal antibody. Since ca1C5 enhanced the function of stimulated canine immune cells, it was suggested that it releases immunosuppression via the CTLA-4 pathway and activates antitumor immunity. Safety and pharmacokinetic studies using experimental dogs showed that ca1C5 demonstrated a certain level of safety and expected pharmacokinetics both alone and in combination with the canine chimeric anti-PD-L1 antibody c4G12. Because ca1C5 showed favorable antitumor efficacy in dogs with recurrent oral malignant melanoma during treatment with c4G12, it was revealed that ca1C5 can be used as a novel immunotherapy agent for canine tumor treatment.

[0127] [Example 2] Feline Anti-CTLA-4 Antibody 1. Introduction The rat anti-canine CTLA-4 monoclonal antibody (clone name: 1C5-E5) showed cross-reactivity with feline CTLA-4, suggesting its potential use as an immunotherapy agent for feline tumors. Therefore, in this example, a feline anti-CTLA-4 antibody fe1C5 was created using the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5, and its biological activity was investigated using cytokine production in a feline peripheral blood mononuclear cell culture system as an indicator.

[0128] 2. Materials and Methods 2.1. Examination of the binding affinity of anti-CTLA-4 antibodies to feline CTLA-4 by surface plasmon resonance (SPR) analysis To produce an expression plasmid for recombinant protein (feline CTLA-4-His) in which polyhistidine (6 × His) is fused to the C-terminus of the extracellular region of feline CTLA-4, PCR was performed using a pMD20 plasmid (Takara Bio Inc.) in which the coding region of feline CTLA-4 (SEQ ID NO: 58, SEQ ID NO: 59) was inserted, with primers (feCTLA-4-His_F and feCTLA-4-His_R) (SEQ ID NO: 98, SEQ ID NO: 99) having recognition sites for restriction enzymes EcoRV-HF (New England Biolabs) and KpnI-HF (New England Biolabs) added to the 5′ end. The obtained PCR products were treated with each restriction enzyme, purified using the FastGene Gel / PCR Extraction Kit (Nippon Genetics Co., Ltd.), and cloned into a pCXN2.1 vector (Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199.) that had undergone similar restriction enzyme treatment. The resulting expression plasmid was purified using the FastGene Xpress Plasmid PLUS Kit (Nippon Genetics Co., Ltd.).

[0129] Next, 7.5 × 10 7Expi293F cells (Thermo Fisher Scientific) were suspended in 25.5 mL of Expi293 Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 30 μg of expression plasmid was introduced using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) to express the target protein. Feline CTLA-4-His was purified from the culture supernatant using TALON Metal Affinity Resin (Takara Bio). After purification, the buffer was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using Amicon Ultra-4 Ultracel-3 (Merck Millipore). The concentration of purified feline CTLA-4-His was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0130] To investigate the binding affinity (avidity) of each antibody to feline CTLA-4 using feline CTLA-4-His, SPR analysis was performed using the Biacore X100 system (GE Healthcare). Anti-His antibodies were immobilized on Sensor Chip CM5 (GE Healthcare) by amine coupling using the His Capture Kit (GE Healthcare). Feline CTLA-4-His was captured on the sensor chip as the ligand, and each antibody, ranging from 20 nM to 2-fold serial dilutions, was used as the analyte. Curve fitting was performed on the obtained sensorgrams using a 1:1 kinetic binding model to determine the binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D The following was calculated. For comparison, the canine CTLA-4-His prepared in [Example 1] was used.

[0131] 2.2. Production of Feline Anti-CTLA-4 Antibodies The complementarity-determining regions (SEQ ID NOs: 1-6) contained in the predicted amino acid sequences (SEQ ID NOs: 29, 31) of the heavy and light chain variable regions of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 were transplanted into the framework regions of the feline immunoglobulin heavy chain variable region sequence (SEQ ID NOs: 60) and the feline immunoglobulin κ chain variable region sequence (SEQ ID NOs: 61) to design the feline anti-CTLA-4 antibody heavy and light chain variable region amino acid sequences (SEQ ID NOs: 62 and 63). The obtained variable region sequences were combined with the feline IgG1a constant region sequence (SEQ ID NOs: 64) and the feline immunoglobulin κ chain constant region sequence (SEQ ID NOs: 65) to form the heavy and light chain sequences (SEQ ID NOs: 66 and 68) of the feline anti-CTLA-4 antibody 1C5-E5 (fe1C5-E5).

[0132] The nucleotide sequences encoding these amino acids were optimized for expression in Chinese hamster-derived cells (SEQ ID NOs. 67 and 69), and artificial genes were synthesized (Eurofins Genomics). Each sequence was inserted into the pCEC2.1 vector (Eurofins Genomics), and the resulting expression plasmid was purified using NucleoBond Xtra Midi (Takara Bio). The mutant feline anti-CTLA-4 antibody (fe1C5-E5-V1 and fe1C5-E5-V2) light chain (SEQ ID NOs. 72 and 74) expression plasmids were prepared by modifying the gene sequence encoding the light chain (SEQ ID NOs. 73 and 75) by overlap PCR using mutation primers (fe1C5L_OL1_F, fe1C5L_OL1_R, fe1C5L_OL2_F, and fe1C5L_OL2_R) (SEQ ID NOs. 100-103), and then inserting the target gene fragment into the pCEC2.1 vector in the same manner. 1.5 × 10⁻⁶ 8ExpiCHO-S cells (Thermo Fisher Scientific) were suspended in 25 mL of ExpiCHO Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 10 μg each of antibody heavy chain and light chain expression plasmids were transfected into ExpiCHO-S cells using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) to express the target antibody. From the resulting culture supernatant, fe1C5-E5, fe1C5-E5-V1, and fe1C5-E5-V2 were purified using Ab-Capcher ExTra (ProteNova). After purification, the solvent was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using PD midiTrap G-25 (GE Healthcare). The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific).

[0133] To confirm the expression and purification of fe1C5-E5, fe1C5-E5-V1, and fe1C5-E5-V2, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Equal volumes of 2 × Laemmli Sample Buffer (BIO-RAD) containing 2-Mercaptoethanol (2-ME) (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the antibody-containing solution and treated at 96°C for 5 minutes (reducing conditions). A sample solution without 2-ME was also prepared (non-reducing conditions). Subsequently, sample proteins were separated by electrophoresis using SuperSep Ace 5-20% (Fujifilm Wako Pure Chemical Industries, Ltd.). Precision Plus Protein Standards all blue (Bio-Rad) was used as the molecular weight marker. After electrophoresis, the gels were stained with a high-sensitivity CBB staining kit (Integrale), and then destained by two passes of permeation in heated distilled water. For comparison, rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 was used.

[0134] 2.3. Establishment of stable expression cells for the therapeutic feline anti-CTLA-4 antibody fe1C5 The heavy and light chain variable regions of fe1C5-E5-V2 (SEQ ID NOs. 62 and 71) were combined with the feline IgG1a constant region sequence (SEQ ID NOs. 64) and the feline immunoglobulin κ chain constant region sequence (SEQ ID NOs. 65) to design the heavy and light chain amino acid sequences (SEQ ID NOs. 66 and 74) of the therapeutic feline anti-CTLA-4 antibody fe1C5. The nucleotide sequences encoding the heavy and light chain amino acids (SEQ ID NOs. 66 and 74) of fe1C5 (fe1C5-E5-V2) were codon-optimized for expression in Chinese hamster-derived cells (SEQ ID NOs. 76 and 77), and artificial gene synthesis (Genscript) was performed by adding recognition sequences for restriction enzymes NotI and SbfI (heavy chain) or AscI and AsiSI (light chain). pDC62c5-U533 vector (Suzuki, Y., Nakagawa, M., Kameda, Y., Konnai, S., Okagawa, T., Maekawa, N., Goto, S., Sajiki, Y., Ohashi, K., Murata, S., Kitahara, Y. and Yamamoto, K. 2020. Novel vector and use thereof. US patent application No. 17 / 054,936.) with restriction enzymes AscI, AsiSI, NotI-HF and SbfI-HF (New England Biolabs) and DNA Ligation Kit<Mighty Mix> The target gene fragment was inserted using (Takara Bio Inc.), and the plasmid was purified using NucleoBond Xtra Midi (Takara Bio Inc.). 1.5 × 10 7CHO-DG44 cells (Thermo Fisher Scientific) were suspended in 30 mL of CD-DG44 medium (Thermo Fisher Scientific) containing 20 mL / L of GlutaMAX-I supplement (Thermo Fisher Scientific) and 18 mL / L of 10% Pluronic F-68 (Thermo Fisher Scientific), and cultured in 125 mL flasks (Corning). 18 μg of expression plasmid was introduced using OptiPRO SFM (Thermo Fisher Scientific) and FreeStyle MAX Reagent (Thermo Fisher Scientific), and after 48 hours, the medium was changed to 10 mL of Dynamis medium (Thermo Fisher Scientific) (hereafter referred to as Dynamis-ACA medium) containing 1% Anti-Clumping Agent (Thermo Fisher Scientific) and 4 mM GlutaMAX-I (Thermo Fisher Scientific). After 14 days, stable expression clones were obtained using the AS ONE Cell Picking System (Furukawa Electric Co., Ltd.) (Yoshimoto, N., Kida, A., Jie, X., Kurokawa, M., Iijima, M., Niimi, T., Maturana, AD, Nikaido, I., Ueda, HR, Tatematsu, K., Tanizawa, K., Kondo, A., Fujii, I. and Kuroda, S. 2013. An automated system for high-throughput single cell-based breeding. Scientific Reports 3:1191.). The established stable expression clones were cultured in Dynamis-ACA medium at a dose of 0.5 × 10⁶. 6The antibodies were suspended at a concentration of cells / mL and cultured with shaking (125 rpm) at 35°C in the presence of 8% CO2 for 17 days. 4 g / L, 4 g / L, and 6 g / L glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) were added on days 3, 5, and 7, respectively, and 3.3% v / v EfficientFeed B+ 3× Supplement (Thermo Fisher Scientific, Inc.) was added on days 3, 5, 7, and 10, respectively. After 17 days of shaking culture, the culture supernatant was collected using Sartoclear Dynamics Lab V (Sartorius, Inc.) and subjected to two-step antibody purification by protein A affinity chromatography and anion exchange chromatography. After purification, the solvent of the antibody solution was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries, Ltd.) using Viva flow 50 (Sartorius, Inc.). Finally, the samples were filtered through 0.45 μm and 0.2 μm filters (Sartorius) and stored at 4°C until use in experiments. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm using a NanoDrop8000 spectrophotometer (Thermo Fisher Scientific).

[0135] 2.4. Examination of the inhibitory activity of anti-CTLA-4 antibodies against CTLA-4 / CD80 binding and CTLA-4 / CD86 binding The inhibitory activity of rat anti-canine CTLA-4 monoclonal antibodies 1C5-E5 and fe1C5 against CTLA-4 / CD80 binding and CTLA-4 / CD86 binding was examined using recombinant proteins (CD80-Ig, CD86-Ig, and CTLA-4-Ig) in which the extracellular domain of each factor was expressed as a C-terminal rabbit IgG Fc fusion protein.

[0136] To create recombinant protein expression plasmids in which rabbit IgG-Fc is fused to the C-terminus of the extracellular region of feline CTLA-4, CD80, or CD86, PCR was performed using pMD20 plasmids (Takara Bio Inc.) containing the coding regions of feline CTLA-4 (SEQ ID NO: 58, SEQ ID NO: 59), feline CD80 (SEQ ID NO: 78, SEQ ID NO: 79), or feline CD86 (SEQ ID NO: 80, SEQ ID NO: 81) as templates, and primers (feCTLA-4-Ig_F, feCTLA-4-Ig_R, feCD80-Ig_F, feCD80-Ig_R, feCD86-Ig_F, feCD86-Ig_R) (SEQ ID NOs: 104-109) with recognition sites for restriction enzymes EcoRV-HF (New England Biolabs) and KpnI-HF (New England Biolabs) added to the 5′ end. The obtained PCR products were treated with each restriction enzyme, purified using the FastGene Gel / PCR Extraction Kit (Genetics Japan), and cloned into a pCXN2.1-Rabbit IgG Fc vector (modified from Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108, 193-199.) that had undergone similar restriction enzyme treatment. The resulting expression plasmid was purified using NucleoBond Xtra Midi (Takara Bio Inc.).

[0137] 7.5×10 7Expi293F cells (Thermo Fisher Scientific) were suspended in 25.5 mL of Expi293 Expression Medium (Thermo Fisher Scientific) and cultured in a 125 mL flask (Corning). 30 μg of each expression plasmid was introduced using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) to express the target protein. Recombinant proteins (CD80-Ig, CD86-Ig, and CTLA-4-Ig) were purified from the resulting culture supernatant using Ab-Capcher ExTra (ProteNova). After purification, the buffer was replaced with PBS pH 7.2 (Fujifilm Wako Pure Chemical Industries) using PD midiTrap G-25 (GE Healthcare). The concentrations of the purified recombinant proteins were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0138] 50 μL each of CD80-Ig and CD86-Ig, diluted to 1 μg / mL, were dispensed into 96-well ELISA plates (Thermo Fisher Scientific) and reacted at 37°C for 30 minutes to solidify the phase. After five washes, 200 μL of PBS containing 0.05% Tween20 (Kanto Chemical Co.) and 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich) was added, and the mixture was blocked at 37°C for 30 minutes. Simultaneously, in a separate 96-well plate (Corning), biotinylated CTLA-4-Ig using the Lightning-Link Rapid Biotin Conjugation Kit (Innova Biosciences) was mixed with each antibody at molar concentrations of 0, 0.1, 0.5, 1, 2, 5, and 10 (antibody / CTLA-4-Ig), and reacted at 37°C for 30 minutes. The final concentration of biotinylated CTLA-4-Ig was 200 pM in the CD80-Ig / CTLA-4-Ig binding assay and 100 pM in the CD86-Ig / CTLA-4-Ig binding assay. Rat IgG was used as the negative control antibody. 2bBD Biosciences and feline IgG (Jackson ImmunoResearch) were used at the same concentration. After washing a 96-well ELISA plate five times, 50 μL of a mixture of antibody and biotinylated CTLA-4-Ig was added to each plate and reacted at 37°C for 30 minutes. After five washes, 50 μL of NeutrAvidin Horseradish Peroxidase Conjugate (Thermo Fisher Scientific), diluted to 1 μg / mL, was added to each plate and reacted at 37°C for 30 minutes. After five washes, 50 μL of TMB one component substrate (Bethyl Laboratories) was added and reacted in the dark, then 50 μL of 0.18 M H2SO4 was added to stop the reaction, and the absorption wavelength was measured at 450 nm using a microplate reader MTP-900 (Corona Electric). All washings were performed using PBS with 0.05% Tween20 (Kanto Chemical). PBS was used to dilute CD80-Ig and CD86-Ig, while PBS containing 0.05% Tween20 (Kanto Chemical Co., Ltd.) and 1% BSA (Sigma-Aldrich Co., Ltd.) was used to dilute biotinylated CTLA-4-Ig and the antibody.

[0139] 2.5. Investigation of the activation effect of feline peripheral blood mononuclear cells (PBMCs) using feline anti-CTLA-4 antibody fe1C5. Feline PBMCs were isolated from heparinized feline peripheral blood by density gradient centrifugation using Percoll (GE Healthcare). The isolated PBMCs were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific), antibiotics (streptomycin 100 μg / mL, penicillin 100 U / mL) (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). A superantigen (Staphylococcal enterotoxin B) (Sigma-Aldrich) was added at a final concentration of 5 μg / mL, and the cells were incubated statically for 3 days at 37°C in the presence of 5% CO2. The production of IL-2, IFN-γ, and TNF-α in the culture supernatant after adding fe1C5 at a final concentration of 10 μg / mL was measured using Feline IL-2 DuoSet ELISA (R&D Systems), Feline IFN-gamma DuoSet ELISA (R&D Systems), and Feline TNF-alpha DuoSet ELISA (R&D Systems). Feline IgG at the same concentration (Jackson ImmunoResearch) was used as a negative control. A microplate reader MTP-900 (Corona Electric Co., Ltd.) was used to measure absorbance.

[0140] 2.6. Statistical Analysis Tukey's multiple comparison test and Wilcoxon's signed-rank test were used for statistical testing. A significance level (p) of less than 0.05 was considered statistically significant.

[0141] 3. Results and Discussion 3.1. Examination of the binding affinity of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 to feline CTLA-4 A comparison of the predicted amino acid sequences of canine CTLA-4 and feline CTLA-4 revealed that their homology was approximately 97%, indicating a high degree of conservation between the two species (Figure 16). From this, it was considered highly likely that 1C5-E5 would also cross-react with feline CTLA-4. Therefore, a recombinant protein (feCTLA-4-His) was created by fusing a 6 × histidine tag to the extracellular region of feline CTLA-4, and the binding affinity (avidity) of 1C5-E5 and feline CTLA-4 was examined by SPR analysis. The binding rate constant (k) was calculated using a 1:1 kinetic binding model. a ), dissociation rate constant (k d ), and the dissociation constant (K D ) are 3.46 ± 0.16 × 10 5 / Ms, 6.24 ± 0.19 × 10 -4 / s, and 1.73 ± 0.14 × 10 -10 The reaction rate constant was M (Figure 17). These reaction rate constants were comparable to those of 1C5-E5 for canine CTLA-4 calculated by similar measurements, indicating that 1C5-E5 has a binding affinity for feline CTLA-4 equivalent to that for canine CTLA-4.

[0142] 3.2. Production of Feline Anti-CTLA-4 Antibody (fe1C5-E5) To create an anti-CTLA-4 antibody with reduced immunogenicity that can be repeatedly administered to cats, a feline anti-CTLA-4 antibody (fe1C5-E5) was designed by transplanting the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 into a cat-derived antibody sequence (Figure 18). An expression vector for fe1C5-E5 was prepared, and transient expression was performed in mammalian cells to obtain a culture supernatant containing fe1C5-E5. Analysis of the purified fe1C5-E5 by SDS-PAGE revealed a band considered to be a heterotetramer at a position slightly smaller than 250 kDa under non-reducing conditions, and bands considered to be heavy chain and light chain at approximately 50 kDa and 25 kDa, respectively, under reducing conditions (Figure 19).

[0143] 3.3. Examination of the binding affinity of feline anti-CTLA-4 antibody fe1C5-E5 to feline CTLA-4 by SPR analysis To examine the binding affinity of fe1C5-E5 to feline CTLA-4, the reaction rate constants for the binding of rat anti-canine CTLA-4 monoclonal antibody 1C5-E5 and fe1C5-E5 to feline CTLA-4 were calculated using SPR analysis with a 1:1 kinetic binding model. Binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D ) were 3.75 ± 0.25 × 10 for rat anti-canine CTLA-4 monoclonal antibody 1C5-E5, respectively. 6 / Ms, 10.1 ± 0.27 × 10 -4 / s, and 2.72 ± 0.24 × 10 -10 M is the value, and for fe1C5-E5, it is 1.97 ± 0.04 × 10⁻⁶ respectively. 6 / Ms, 10.7 ± 0.87 × 10 -4 / s, and 5.41 ± 0.39 × 10 -10 The result was M (Figure 20). The dissociation constant of fe1C5-E5 was approximately twice as high as that of the original 1C5-E5, suggesting that the binding affinity to feline CTLA-4 decreased in fe1C5-E5 as the antibody became feline.

[0144] 3.4. Production of mutant feline anti-CTLA-4 antibodies (fe1C5-E5-V1 and fe1C5-E5-V2) To restore the decreased binding affinity of fe1C5-E5 to feline CTLA-4 associated with feline mutation, mutant feline anti-CTLA-4 antibodies (fe1C5-E5-V1 and fe1C5-E5-V2) were produced by introducing amino acid substitutions into the fe1C5-E5 light chain variable region. The amino acid sequences of the light chain variable regions of fe1C5-E5-V1 and fe1C5-E5-V2 are shown in SEQ ID NOs. 70 and 71, respectively. Expression vectors for fe1C5-E5-V1 and fe1C5-E5-V2 were prepared, and transient expression in mammalian cells was performed to obtain culture supernatants containing fe1C5-E5-V1 and fe1C5-E5-V2. After purification, fe1C5-E5-V1 and fe1C5-E5-V2 were analyzed by SDS-PAGE and showed electrophoretic patterns similar to those of fe1C5-E5 (Figure 19). Therefore, fe1C5-E5-V1 and fe1C5-E5-V2 were subjected to the following functional tests.

[0145] 3.5. Investigation of the binding affinity of mutant feline anti-CTLA-4 antibodies fe1C5-E5-V1 and fe1C5-E5-V2 to feline CTLA-4 using SPR analysis. To investigate the binding affinity of fe1C5-E5-V1 and fe1C5-E5-V2 to feline CTLA-4, the reaction rate constants for binding between fe1C5-E5-V1 and fe1C5-E5-V2 and feline CTLA-4 were calculated using SPR analysis with a 1:1 kinetic binding model. Binding rate constant (k a ), dissociation rate constant (k d ), and the dissociation constant (K D ) are 3.42 ± 0.04 × 10 for fe1C5-E5-V1 respectively. 6 / Ms, 8.38 ± 0.53 × 10 -4 / s, and 2.33 ± 0.17 × 10 -10 M is the value, and for fe1C5-E5-V2, it is 4.38 ± 0.03 × 10⁻¹⁰. 6 / Ms, 8.82 ± 0.44 × 10 -4 / s, and 2.01 ± 0.10 × 10 -10 The result was M (Figure 20). Compared to fe1C5-E5, the dissociation constants of fe1C5-E5-V1 and fe1C5-E5-V2 were about twice as low, suggesting that their binding affinity to feline CTLA-4 recovered to roughly the same level as that of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5. Furthermore, comparing fe1C5-E5-V1 and fe1C5-E5-V2, the dissociation constant of fe1C5-E5-V2 was even lower, suggesting that fe1C5-E5-V2 had a higher binding affinity to feline CTLA-4.

[0146] 3.6. Establishment of stable high-expression cells of the therapeutic feline anti-CTLA-4 antibody fe1C5 Based on the above results, among the feline anti-CTLA-4 antibodies produced (fe1C5-E5, fe1C5-E5-V1, and fe1C5-E5-V2), fe1C5-E5-V2 was considered the most suitable candidate for therapeutic antibody. A large quantity of the antibody is needed to investigate safety through administration studies using healthy cats and to investigate therapeutic efficacy through clinical trials in cats with tumors. Therefore, we established stable, high-expression cell clones that highly express the therapeutic feline anti-CTLA-4 antibody fe1C5 (fe1C5-E5-V2) using the pDC62c5-U533 / CHO-DG44 expression system (Suzuki, Y., Nakagawa, M., Kameda, Y., Konnai, S., Okagawa, T., Maekawa, N., Goto, S., Sajiki, Y., Ohashi, K., Murata, S., Kitahara, Y. and Yamamoto, K. 2020. Novel vector and use thereof. US patent application No. 17 / 054,936.). After 17 days of production culture, fe1C5 was purified from the culture supernatant, and the purified fe1C5 was analyzed by SDS-PAGE. Similar to fe1C5-E5-V2 produced using a transient expression system, the observed band positions were in general agreement with the molecular weight calculated from the predicted amino acid sequence (Figure 21). Therefore, fe1C5 was subjected to the following functional tests.

[0147] 3.7. Examination of the binding affinity of the felineized anti-CTLA-4 antibody fe1C5 to feline CTLA-4 To examine the binding affinity of fe1C5 to feline CTLA-4, the reaction rate constants in the binding of fe1C5 and feline CTLA-4 were calculated using SPR analysis with a 1:1 kinetic binding model. The association rate constant (k a ), the dissociation rate constant (k d ), and the dissociation constant (K D ) were 4.21 ± 0.05 × 10 6 / Ms, 8.71 ± 0.20 × 10 -4 / s, and 2.07 ± 0.07 × 10 -10 M, respectively (Figure 22), and were generally comparable to those of fe1C5-E5-V2 prepared by a transient expression system and the rat anti-dog CTLA-4 monoclonal antibody 1C5-E5 (Figure 20).

[0148] 3.8. Examination of the inhibitory ability of the felineized anti-CTLA-4 antibody fe1C5 against CTLA-4 / CD80 binding and CTLA-4 / CD86 binding Next, the inhibitory ability of fe1C5 against CTLA-4 / CD80 binding or CTLA-4 / CD86 binding was examined. A solution in which biotinylated feline CTLA-4 recombinant protein (CTLA-4-Ig) and each anti-CTLA-4 antibody were pre-reacted at various molar concentration ratios was added to an ELISA plate immobilized with feline CD80 recombinant protein (CD80-Ig) or feline CD86 recombinant protein (CD86-Ig), and the CTLA-4-Ig bound to CD80-Ig or CD86-Ig was detected by a color reaction using enzyme-labeled avidin. fe1C5 inhibited CTLA-4 / CD80 binding and CTLA-4 / CD86 binding, and its inhibitory effect was comparable to that of the rat anti-dog CTLA-4 monoclonal antibody 1C5-E5 (Figure 23).

[0149] 3.9. Investigation of the effect of feline anti-CTLA-4 antibody fe1C5 on feline PBMC activation To investigate the immune-activating effect of fe1C5, we examined its effect on cytokine production in feline PBMCs stimulated and cultured for 3 days in the presence of a superantigen. When the concentrations of IL-2, IFN-γ, and TNF-α in the culture supernatant were quantified by ELISA, the production of IL-2 and TNF-α was significantly increased when fe1C5 was added compared to when feline IgG, which was used as a negative control antibody, was added (Figure 24). These results suggest that fe1C5 has the effect of enhancing the activation of feline immune cells.

[0150] 4. Conclusion The felineized anti-CTLA-4 monoclonal antibody fe1C5, created using the complementarity-determining region of the rat anti-canine CTLA-4 monoclonal antibody 1C5-E5, exhibited similar binding affinity to feline CTLA-4 and inhibitory effects on feline CTLA-4 / CD80 and CTLA-4 / CD86 binding as the original rat monoclonal antibody. Since fe1C5 enhanced the function of stimulated feline immune cells, it was suggested that it has the effect of reversing immunosuppression by the CTLA-4 pathway and activating antitumor immunity.

[0151] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0152] The anti-CTLA-4 antibody of the present invention can be used for the prevention and / or treatment of cancer and infectious diseases in animals.

[0153]

Claims

(a) A H chain having a variable H chain region including CDR-H1 containing the amino acid sequence NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (b) An anti-CTLA-4 antibody or its antigen-binding fragment, comprising an L chain having an L chain variable region, comprising CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

20. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 1, wherein the H chain variable region and the L chain variable region are derived from rats. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the H chain and L chain each have a constant H chain region and a constant L chain region of an animal antibody other than rat. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 3, wherein the animal other than rat is a dog. An anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 29 and the L chain variable region comprises the amino acid sequence of SEQ ID NO:

31. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 1, which is canine-modified. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 6, wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 34 and the L chain variable region comprises the amino acid sequence of SEQ ID NO:

35. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 6, wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 34, and the L chain variable region comprises the amino acid sequence of SEQ ID NO: 50 or 51. An anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the H chain constant region comprises the amino acid sequence of SEQ ID NO: 36 or 52, and the L chain constant region comprises the amino acid sequence of SEQ ID NO: 37 or 53. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 1, comprising an H chain having a variable H chain region containing the amino acid sequence of SEQ ID NO: 34 and a constant H chain region of SEQ ID NO: 52, and an L chain having a variable L chain region containing the amino acid sequence of SEQ ID NO: 51 and a constant L chain region of SEQ ID NO:

53. The anti-CTLA-4 antibody according to claim 1, or its antigen-binding fragment, which is felineized. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 11, wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 62 and the L chain variable region comprises the amino acid sequence of SEQ ID NO:

63. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 11, wherein the H chain variable region comprises the amino acid sequence of SEQ ID NO: 62, and the L chain variable region comprises the amino acid sequence of SEQ ID NO: 70 or 71. An anti-CTLA-4 antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, comprising an H chain having an H chain variable region containing the amino acid sequence of SEQ ID NO: 62 and an H chain constant region containing the amino acid sequence of SEQ ID NO: 64, and an L chain having an L chain variable region containing the amino acid sequence of SEQ ID NO: 63, 70, or 71 and an L chain constant region containing the amino acid sequence of SEQ ID NO:

65. The anti-CTLA-4 antibody or antigen-binding fragment thereof according to claim 11, comprising an H chain having a variable H chain region containing the amino acid sequence of SEQ ID NO: 62 and a constant H chain region containing the amino acid sequence of SEQ ID NO: 64, and an L chain having a variable L chain region containing the amino acid sequence of SEQ ID NO: 71 and a constant L chain region containing the amino acid sequence of SEQ ID NO:

65. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15. A vector comprising the polynucleotide described in claim 16. Host cells transformed with the vector according to claim 17. A method for producing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in claim 18 and collecting an anti-CTLA-4 antibody or an antigen-binding fragment thereof from the culture. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as an active ingredient, according to any one of claims 1 to 15. The pharmaceutical composition according to claim 20 for the prevention and / or treatment of cancer and / or infectious diseases. The pharmaceutical composition according to claim 21, wherein the cancer and / or infectious disease is selected from the group consisting of neoplastic diseases, leukemia, viral diseases, prion diseases, bacterial diseases, mycoplasma diseases, rickettsial diseases, chlamydia diseases, fungal diseases and protozoal diseases. A pharmaceutical composition according to any one of claims 20 to 22, which is administered before, after, or simultaneously with the administration of a PD-1 / PD-L1 targeting inhibitor. The pharmaceutical composition according to claim 23, wherein the inhibitor targeting PD-1 / PD-L1 is an antibody or an antigen-binding fragment thereof. The pharmaceutical composition according to claim 24, wherein the antibody is an anti-PD-1 antibody or its antigen-binding fragment, or an anti-PD-L1 antibody or its antigen-binding fragment. (a1) A polynucleotide encoding an H chain or an antigen-binding fragment thereof having an H chain variable region, including CDR-H1 containing the amino acid sequence of NYY, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (b1) A combination of a polynucleotide encoding an L-chain variable region or an antigen-binding fragment thereof, comprising CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

20. (a2) A vector incorporating polynucleotides encoding an H chain or an antigen-binding fragment thereof having an H chain variable region, including CDR-H1 containing the NYY amino acid sequence, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (b2) A combination of a vector incorporating polynucleotides encoding an L-chain variable region or an antigen-binding fragment thereof, including CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of Sequence ID No.

20. (a2) A vector incorporating polynucleotides encoding an H chain or an antigen-binding fragment thereof having an H chain variable region, including CDR-H1 containing the NYY amino acid sequence, CDR-H2 containing the amino acid sequence of SEQ ID NO: 10, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, (b2) Host cells transformed by a combination of a vector incorporating polynucleotides encoding an L-chain variable region or an antigen-binding fragment thereof, including CDR-L1 containing the amino acid sequence of SKY, CDR-L2 containing the amino acid sequence of SGS, and CDR-L3 containing the amino acid sequence of Sequence ID No.

20. A method for producing an anti-CTLA-4 antibody or an antigen-binding fragment thereof, comprising culturing the host cells described in claim 28 and collecting an anti-CTLA-4 antibody or an antigen-binding fragment thereof from the culture.

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