Human CD153-targeting antibody and chimeric antigen receptor

Abstract

The present application provides an anti-human CD153 monoclonal antibody that specifically binds to human CD153 and inhibits an intermolecular interaction between human CD153 and CD30, or a binding fragment thereof. The present application also provides: a chimeric antigen receptor including an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain has the same heavy-chain hypervariable region and the same light-chain hypervariable region as those in the monoclonal antibody according to the present application; and a CAR-T cell capable of expressing the chimeric antigen receptor. The monoclonal antibody and the binding fragment thereof and the CAR-T cell according to the present application are useful for the treatment or prevention of diseases associated with CD153, such as multiple sclerosis.

Description

Antibodies and chimeric antigen receptors targeting human CD153

[0001] The present disclosure relates to antibodies and chimeric antigen receptors that target human CD153.

[0002] It is known that a population of T cells called senescence-associated T cells (SA-T cells), which are predisposing factors for the onset and exacerbation of inflammatory diseases, increases with aging. Unlike normal T cells, SA-T cells are unable to exhibit T cell antigen receptor (TCR)-mediated proliferation and activation responses, while exhibiting the characteristics of senescent cells, capable of secreting large amounts of inflammatory factors. A vaccine containing a specific partial amino acid sequence of CD153 as an immunogen has been proposed as a vaccine targeting SA-T cells for the prevention or treatment of glucose metabolism disorders (Patent Document 1).

[0003] Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, characterized by the temporal and spatial multiplication of lesions. A diagnosis is usually confirmed by demonstrating the temporal and spatial multiplication of lesions through a detailed medical history and a longitudinal neurological examination, and by ruling out other diseases. The cause of MS is still unclear, but it is thought that lymphocytes and macrophages infiltrate the lesions, leading to demyelination due to inflammation mediated by an autoimmune mechanism. Furthermore, due to racial differences, genetic and environmental factors have been suggested to be involved, but the exact cause remains unclear.

[0004] Treatment for MS consists of treatment for acute exacerbations, treatment to prevent relapse and progression, symptomatic treatment for the acute and chronic phases, and rehabilitation, but there is currently no reliable method to prevent relapse or progression of MS. MS affects young adults and has a long-term course of relapse and remission. Relatively severe damage remains in the optic nerve, spinal cord, and cerebellum, and in many cases, active daily living (ADL) is significantly reduced. There is a long awaited need to elucidate the mechanism of MS and develop highly effective antibodies and vaccines that will be useful in treatment.

[0005] Patent Document 2 proposes the use of CD30L, ie, an antibody specific to CD153, for the treatment of multiple sclerosis, but the effect is limited.

[0006] CAR-T cells are peripheral T cells forced to express a CAR consisting of a single-chain antibody (scFv) sequence and an activation signaling domain, and have a high ability to kill target cells. Since intravenously administered CAR-T cells can pass through the blood-brain barrier (BBB) ​​and reach the central nervous system, and local administration of CAR-T cells is also possible, it has been suggested that they are effective in eliminating cells within the central nervous system (Non-Patent Document 1).

[0007] WO2020 / 096046 U.S. Patent No. 6,652,854

[0008] Huang et al., 2023, Mol Cancer 22:22. Patent and non-patent literature is incorporated herein by reference.

[0009] The present disclosure aims to provide a method for treating multiple sclerosis. The present disclosure also aims to provide an anti-human CD153 monoclonal antibody or a binding fragment thereof that specifically binds to human CD153 and inhibits the intermolecular interaction between CD153 and CD30.

[0010] Another object of the present application is to provide a chimeric antigen receptor (CAR) that can specifically bind to human CD153 and eliminate or kill cells that express human CD153, as well as CAR-T cells that express the CAR.

[0011] The present disclosure provides an anti-human CD153 monoclonal antibody, or a binding fragment thereof, that specifically binds to human CD153 and inhibits the intermolecular interaction between CD153 and CD30. Examples of anti-human CD153 monoclonal antibodies include antibodies having the following heavy chain complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3) and light chain complementarity-determining regions (CDR-L1, CDR-L2, CDR-L3).

[0012] The anti-human CD153 monoclonal antibodies of the present disclosure are useful for treating and preventing diseases in which CD153 is involved in a subject.

[0013] The present disclosure also provides a chimeric antigen receptor (CAR) capable of specifically binding to CD 153. The chimeric antigen receptor of the present disclosure comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain comprises a single-chain antibody comprising the heavy chain complementarity-determining region and the light chain complementarity-determining region of human CD153 shown in Table 1.

[0014] The present disclosure further provides CAR-T cells that express such a CAR and have antigen-specific killer activity. The CAR-T cells of the present disclosure are useful for treating and preventing diseases in which CD153 is involved in a subject.

[0015] The present disclosure also provides a method for treating or preventing a disease involving CD153 in a subject, comprising administering to a subject in need of treatment an effective amount of an anti-human CD153 monoclonal antibody or binding fragment thereof provided by the present disclosure.

[0016] The present disclosure also provides a method for treating or preventing a disease involving CD153 in a subject, comprising administering to the subject in need of treatment a CAR-T cell of the present disclosure.

[0017] The present disclosure also provides use of an anti-human CD153 monoclonal antibody or binding fragment thereof provided by the present disclosure for the manufacture of a medicament for treating or preventing a disease involving CD153 in a subject.

[0018] The present disclosure also provides use of a CAR-T cell of the present disclosure for the manufacture of a medicament for the treatment or prevention of a disease involving CD153 in a subject.

[0019] Schematic diagram of a multiple sclerosis model of a reference example. Graph showing the results of a reference example. Reactivity of each anti-human monoclonal antibody obtained in Example 1 to human CD153. The average value of two wells is shown. Reactivity of each anti-human monoclonal antibody obtained in Example 1 to human CD153. Measurement results at an antibody concentration of 10 μg / ml. Reactivity of each anti-human monoclonal antibody obtained in Example 1 to mouse CD153. The average value of two wells is shown. Reactivity of each anti-human monoclonal antibody obtained in Example 1 to mouse CD153. The average value of two wells is shown. Reactivity of each anti-human monoclonal antibody obtained in Example 1 to mouse CD153. Measurement results at an antibody concentration of 10 μg / ml. Inhibitory effect of each anti-human monoclonal antibody obtained in Example 1 on the human CD153 / CD30 intermolecular interaction. Inhibitory effect of each anti-human monoclonal antibody obtained in Example 1 on the human CD153 / CD30 intermolecular interaction. Measurement results at an antibody concentration of 10 μg / ml. Reactivity of each anti-human monoclonal antibody obtained in Example 1 against canine and feline CD153 (Example 3). Killing effect of mouse CD153-expressing cells by mCD153-CAR-T cells specific to mouse CD153 obtained in Example 4 (Example 5).

[0020] As used herein and in the claims, "effective amount" refers to the amount of an agent required to cure a disease or ameliorate the symptoms of a disease in an untreated subject. The amount of active compound used in practicing the present invention to prevent or treat a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0021] As used herein and in the claims, "subject" means mammals, including but not limited to humans and non-human mammals, such as cows, horses, dogs, sheep, and cats.

[0022] As used in this specification and claims, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disease and / or its associated symptoms.

[0023] As used in this specification and claims, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like, also include reducing the likelihood of developing a disease in a subject who does not have symptoms of the disease but is at risk of or susceptible to developing the disease, as well as inhibiting the progression of symptoms that have already developed.

[0024] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates, and, as known to those skilled in the art, includes any aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride and Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, infusion and nutritional supplements, such materials, and combinations thereof. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0025] The present disclosure provides an anti-human CD153 monoclonal antibody, or a binding fragment thereof, that specifically binds to human CD153 and inhibits the intermolecular interaction between CD153 and CD30. The present disclosure also provides a pharmaceutical composition comprising the monoclonal antibody or the binding fragment thereof.

[0026] The monoclonal antibodies or binding fragments thereof provided in the present disclosure are useful for treating diseases in which CD153 is involved in a subject. Examples of diseases in which CD153 may be involved include central nervous system inflammatory demyelinating diseases including multiple sclerosis, neuromyelitis optica spectrum disorder (NMOSD), MOG antibody-associated disease (MOGAD), and Baro concentric sclerosis (BCS), peripheral nervous system inflammatory demyelinating diseases including chronic inflammatory demyelinating polyneuropathy (CIDP), autoimmune neuropathies including Fisher syndrome (MFS), autoimmune rheumatic diseases including rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, and ankylosing spondylitis, inflammatory bowel diseases including ulcerative colitis and Crohn's disease, metabolic diseases including diabetes, e.g., type 1 diabetes and type 2 diabetes, and chronic nephritis and renal failure, e.g., age-related chronic renal failure.

[0027] It has been confirmed that monoclonal antibodies having the CDR combinations shown in Table 1 provided in the present disclosure bind not only to human CD153 but also to feline and canine CD153. Therefore, the anti-human CD153 monoclonal antibodies of the present disclosure, or binding fragments thereof, can also be used to treat diseases involving CD153 in canine or feline subjects.

[0028] The monoclonal antibodies or binding fragments thereof provided in the present disclosure are particularly useful for treating multiple sclerosis, which may include, but is not limited to, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, and clinically isolated syndrome.

[0029] As used herein, an "antibody" is an immunoglobulin polypeptide molecule that binds to an antigen. Naturally occurring full-length antibodies are immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The amino-terminal portion of each chain contains a variable region of about 100 to about 110 amino acids primarily responsible for antigen recognition via the complementarity-determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0030] The CDRs are interspersed with more conserved regions called frame regions (FRs). Each light chain variable region (LCVR, also known as VL) and heavy chain variable region (HCVR, also known as VH) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "CDR-L1, CDR-L2, and CDR-L3," and the three CDRs of the heavy chain are referred to as "CDR-H1, CDR-H2, and CDR-H3." The CDRs contain most of the residues that form specific interactions with the antigen.

[0031] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. The mAbs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. An intact mAb contains two heavy chains and two light chains. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology, such as CDR grafting, or a combination of such techniques or other techniques known in the art.

[0032] In the present disclosure, the phrase "humanized antibody" refers to a monoclonal antibody that has a combination of the heavy and light chain CDRs provided in the present disclosure and that has been generated and / or engineered so that the framework regions surrounding the CDRs have framework sequences that are substantially similar or identical to those of human variants. Antibody humanization is well known in the art. The humanized antibodies provided in the present disclosure may be intentionally modified compared to the native sequence, for example, in the constant region, to alter effector or biofunctional properties, or biophysical properties, particularly stability, developability, and / or solubility.

[0033] In the present disclosure, "binding fragments" of antibodies include, for example, Fab fragments, Fab' fragments, F(ab') ２The term "antibody" refers to fragments that bind to the same antigen as the antibody and exhibit functional activity similar to that of the antibody.

[0034] A "framework region" or "framework sequence" refers to any one of framework regions 1-4. Engineered humanized antibodies and antigen-binding fragments thereof encompassed by the present disclosure include molecules in which any one or more of framework regions 1-4 are substantially or fully human (i.e., any possible combination of individual substantially or fully human framework regions 1-4 is present). For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc., are substantially or fully human. A substantially human framework is one that has at least about 80% sequence identity to a known human germline framework sequence. Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) or from "The 20 Immunoglobulin Facts Book" by Marie-Paule Lefranc and Gerard Lefranc (Academic Press, 2001, ISBN 012441351).

[0035] In this disclosure, the terms "caninized antibody" and "feline antibody" refer to monoclonal antibodies that have the same heavy and light chain CDR combinations provided herein as the humanized antibodies described above, but that have been generated and / or engineered so that the framework regions surrounding the CDRs have framework sequences that are substantially similar to or identical to the framework sequences of canine or feline variants. In the case of caninized or feline antibodies, the binding fragments, framework regions, and framework sequences are those obtained by replacing "human" with "canine" or "feline" in the description of humanized antibodies.

[0036] The sequences of CDRs can be defined by various methods, including the Kabat, Chothia, AbM, contact, and IMGT definitions. Unless expressly indicated otherwise, the CDRs in this disclosure are identified by the Kabat definition.

[0037] The present disclosure also provides polynucleotides encoding the monoclonal antibodies or antigen-binding fragments thereof of the present disclosure, as well as expression vectors comprising such polynucleotides.

[0038] In another aspect, the present disclosure provides a chimeric antigen receptor capable of specifically binding to human CD153.

[0039] A "chimeric antigen receptor (CAR)" is a polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. The extracellular antigen-binding domain of the CAR of the present disclosure is preferably an scFv sequence having heavy chain variable regions and light chain variable regions comprising each CDR of the anti-human CD153 antibody shown in Table 1 provided herein.

[0040] An "scFv" is a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, in which the light and heavy chain variable regions are closely linked by a linker, and is an antibody fragment that can be expressed as a single-chain polypeptide. An scFv retains the specificity of the antibody from which it is derived. An scFv may have the light chain variable region and the heavy chain variable region linked to the N-terminal and C-terminal ends of the polypeptide via the linker in either order.

[0041] The extracellular antigen-binding domain may be linked to the transmembrane domain via a hinge sequence. The intracellular signaling domain comprises a functional signaling domain, which may be, for example, the intracellular domain of CD3ζ. Optionally, the antigen-binding domain may further comprise one or more costimulatory factor intracellular domains that provide costimulation of T cells when the antigen-binding domain binds to an antigen. Examples of costimulatory factor intracellular domains include those of CD137, CD27, ICOS, and CD28.

[0042] The present disclosure provides a CAR and a polynucleotide encoding the CAR. The present disclosure further provides a vector comprising the polynucleotide encoding the CAR.

[0043] The present disclosure further provides CAR-T cells that express the CAR of the present disclosure and have antigen-specific killer activity. CAR-T cells can be obtained by expressing the CAR in T cells isolated from the periphery or T cells induced to differentiate from pluripotent stem cells using a vector containing a polynucleotide encoding the CAR.

[0044] Based on experiments using gene-deficient mice, the present inventors have reported that the age-related increase in SA-T cells is related to a unique functional interaction between the CD153 molecule expressed on SA-T cells and the CD30 molecule expressed on a very small number of autoreactive B cells. The interaction between CD153 and CD30 restores TCR-mediated proliferation and activation in SA-T cells, while autoreactive B cells proliferate, differentiate, and produce autoantibodies. In other words, the bidirectional signaling between CD153 and CD30 mutually stimulates SA-T cells and autoreactive B cells, resulting in their activation and proliferation. When mice were administered an antibody (17D) that specifically binds to CD153 and blocks the CD153-CD30 interaction, it was confirmed that the age-related increase in SA-T cells and the associated increase in autoantibodies and inflammatory factors were significantly suppressed.

[0045] Furthermore, it is known that a significant increase in SA-T cells is observed early on in model mice that develop systemic lupus erythematosus (SLE) and die from renal failure. It was confirmed that administering the 17D antibody to such model mice resulted in a strong prevention of the onset of SLE pathology and a therapeutic effect after onset, as well as a clear improvement in survival rate (Fukushima et al., 2022, Cell Reports 40, 111373, the contents of which are incorporated herein by reference).

[0046] The present inventors used the mouse anti-mouse CD153 monoclonal antibody 17D to confirm the effect of this antibody on an animal model of multiple sclerosis. There have been no reports on the relationship between an increase in SA-T cells and multiple sclerosis.

[0047] In the present disclosure, examples of anti-human CD153 antibodies that specifically bind to human CD153 and inhibit the intermolecular interaction between CD153 and CD30 include antibodies having the CDRs shown in Table 1 above. The antibodies of the present disclosure may be humanized, caninized, or felineized by conventional methods. Examples of such antibodies also include antibodies that compete with anti-human CD153 monoclonal antibodies having the above CDRs for binding to human CD153, and humanized, caninized, or felineized antibodies thereof.

[0048] In the present disclosure, examples of "anti-human CD153 antibodies that specifically bind to human CD153 and inhibit the intermolecular interaction between human CD153 and CD30" also include other antibodies that have the ability to bind to the epitope recognized by the anti-human CD153 monoclonal antibody having the above-mentioned CDRs, and humanized, caninized, or feline antibodies thereof.

[0049] The antibodies or binding fragments of the present disclosure may be provided as multispecific antibodies or fragments thereof that contain, in addition to a portion that specifically binds to human CD153, a portion that binds to a different antigen.

[0050] The antibody or antibody fragment of the present disclosure may be conjugated to a drug. The drug may be a small molecule compound, a peptide, a polynucleotide, or the like.

[0051] The antibody or binding fragment thereof provided in this embodiment is provided as a pharmaceutical composition, for example, a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier. An effective amount of the pharmaceutical composition is administered to a subject having or suspected of having a disease in which CD153 is involved, such as multiple sclerosis. The pharmaceutical composition can be administered to a subject by intravenous (IV) administration (e.g., by injection or infusion), intramuscular administration, subcutaneous administration (SC) administration, parenteral administration, intrathecal administration, or epidermal administration.

[0052] The CAR-T cells provided in the present disclosure may be provided as a pharmaceutical composition together with a pharmaceutically acceptable carrier. Similar to the anti-CD153 antibodies of the present disclosure, an effective amount of the pharmaceutical composition is administered to a subject having a disease in which CD153 is involved, such as multiple sclerosis, or a subject suspected of having such a disease. The pharmaceutical composition is administered intravenously (IV) (e.g., by injection or infusion). Reference example

[0053] Effect of mouse anti-mouse CD153 monoclonal antibody 17D on multiple sclerosis models Mouse anti-mouse CD153 monoclonal antibody 17D specifically binds to a mouse antigen, and its epitope is predicted to be located at amino acid positions 154-177 and 197-217 in the extracellular region of mouse CD153. Using mice induced with experimental autoimmune encephalomyelitis (EAE), an animal experimental model of multiple sclerosis, we confirmed that mouse anti-mouse CD153 monoclonal antibody 17D is effective in suppressing the onset and exacerbation of multiple sclerosis.

[0054] The heavy and light chain sequences of the mouse anti-mouse CD153 monoclonal antibody 17D are as follows: Heavy chain: SEQ ID NO: 7 Light chain: SEQ ID NO: 8

[0055] The sequences of the heavy and light chain CDRs of the mouse anti-mouse CD153 monoclonal antibody 17D (hereinafter referred to as 17D) predicted based on the Kabat system are as follows:

[0056] Materials and Methods: Thirteen-week-old female C57BL / 6J mice (n = 10) were immunized subcutaneously (sc) with 200 μg of the MOG35-55 peptide (MEVGWYRSPFSRVHLYRNGK) emulsified in complete Freund's adjuvant (CFA) containing 500 μg of Mycobacterium tuberculosis H37Ra. Additionally, 500 ng of pertussis toxin was administered intraperitoneally on the day of immunization and two days later. Either 100 μl (500 μg) of 17D antibody (n = 5 mice) or 100 μl of PBS (n = 5 mice) was administered intraperitoneally three times a week, starting the day before immunization.

[0057] Clinical signs of EAE were evaluated daily according to the following criteria: 0, normal; 0.5, mild tail droop; 1.0, complete tail droop; 1.5, complete tail droop and mild hindlimb weakness; 2.0, complete tail droop and moderate hindlimb weakness; 2.5, complete tail droop and severe hindlimb weakness; 3.0, complete tail droop and complete paralysis of both hindlimbs; 3.5, complete tail droop, complete paralysis of both hindlimbs, and inability to right itself when placed on its side or both hindlimbs held together on one side of the body; 4.0, complete tail droop, complete paralysis of both hindlimbs, and partial paralysis of forelimbs; 4.5, complete tail droop, complete paralysis of both hindlimbs, partial paralysis of forelimbs, and barely mobile. 5, death or euthanasia due to severe paralysis. The scoring schedule is summarized in Figure 1.

[0058] Statistical analysis: Statistical analysis was performed using GraphPad Prism 9. Comparisons between two groups were performed using the nonparametric two-tailed Mann-Whitney U test. P < 0.05 was considered statistically significant. *; P < 0.05 and **; P < 0.01.

[0059] Results The results are shown in Figure 2. All five mice administered PBS developed EAE around day 13 after MOG / CFA immunization, with the highest average score of 1.5 on days 18-19. In contrast, the four mice administered 17D showed no signs of EAE throughout the experimental period. One mouse administered 17D developed very mild EAE (score 0.5) but recovered within three days.

[0060] EAE incidence: PBS: 5 / 5 (maximum score 2.0) 17D: 1 / 5 (maximum score 0.5) The mouse anti-mouse CD153 antibody 17D prevented the onset or progression of EAE.

[0061] The present inventors also examined the localization of SA-T cells in central nervous system tissue sections from EAE-affected mice obtained using the same method described above. The results confirmed that PD-1-positive helper T cells, including SA-T cells not observed in healthy control mice, were present in the central nervous system proximal to the meninges or infiltrated into the parenchyma (data not shown). Combined with the findings of Fukushima et al., 2022, Cell Reports 40, 111373, in which the mouse anti-mouse CD153 monoclonal antibody 17D inhibited the increase in SA-T cells, these findings demonstrate that an increase in SA-T cells is associated with multiple sclerosis, that the increase in SA-T cells can be inhibited by anti-CD153 antibodies, and that anti-CD153 antibodies are therefore effective in treating multiple sclerosis.

[0062] Obtaining anti-human CD153 monoclonal antibodies (1) Preparation of recombinant human (rh) CD153 protein. The expression vector for the antigen, rhCD153 protein, was constructed by inserting a gene sequence containing a trimerization motif (SP-D) and an 8xHis-tag for purification into p3xFLAG-CMV-9. The constructed vector was transfected into FreeStyle™ 293-F cells and cultured with rotation in an incubator at 37°C and 8% CO2 for 3-4 days. The expressed protein in the culture supernatant was recovered using an affinity purification resin with affinity for the His-tag and then purified by gel filtration chromatography.

[0063] (2) Immunization: Purified rhCD153 (100 μg) was mixed with Freund's complete adjuvant to form an emulsion and administered subcutaneously to 8-week-old CD153-KO B6 mice (female). Three, six, and eight weeks later, rhCD153 (without adjuvant) was administered intraperitoneally (boost). The mice were sacrificed 3 days after the final boost.

[0064] (3) Hybridoma Generation and Screening. Spleen cells obtained from the harvested spleens were fused with myeloma cells SP2 / 0 using PEG1500 and cultured in a 96-well plate using HAT selection. Culture supernatants were collected from wells where colonies formed, and the production of anti-human CD153 antibodies was confirmed by ELISA (primary screening). Cells from wells that showed a positive reaction were cloned by limiting dilution to obtain hybridomas. The hybridoma culture supernatants were then added to hCD153-expressing 293H cells and incubated at room temperature for 30 minutes. After washing, the cells were incubated with Alexa 488-labeled anti-mouse IgG for 30 minutes at room temperature, and clones that showed positive Alexa 488 fluorescence were selected by flow cytometry analysis (secondary screening). Antibodies were purified from the hybridoma culture supernatants of three clones selected by screening (2H7-9, 3A2-5, and 13H9-13) using Protein A. Hereinafter, each purified antibody will be identified by its hybridoma clone name (2H7-9, 3A2-5, 13H9-13).

[0065] (4) Evaluation of Purified Antibody Reactivity The reactivity of purified antibodies was analyzed by flow cytometry. Four-fold serial dilutions of purified antibody solutions (160 μg / ml to 0.00977 μg / ml) and mouse serum IgG (negative control) were added to 293T cells transiently expressing human CD153 and incubated for 20 minutes at 4°C. After washing, the cells were incubated with R-phycoerythrin-labeled anti-mouse IgG for 20 minutes at 4°C, and the R-phycoerythrin fluorescence intensity was compared between clones. The results are shown in Figures 3 and 4.

[0066] (5) Reactivity of purified anti-human CD153 antibodies to mouse CD153 The cross-reactivity of each antibody to mouse CD153 was examined in the same manner as in (4) above, except that EL4 cells stably expressing mouse CD153 were used. The anti-mouse CD153 monoclonal antibody 17D was used as a positive control. The results are shown in Figures 5 to 7. None of the purified antibodies 2H7-9, 3A2-5, or 13H9-13 reacted with mouse CD153.

[0067] (6) Evaluation of the inhibitory effect of purified anti-human CD153 antibodies on the human CD153 / CD30 intermolecular interaction. The inhibitory effect of purified anti-human CD153 antibodies on the intermolecular interaction was analyzed by flow cytometry. Dilutions of purified antibody solution (40, 10, and 2.5 μg / ml) and mouse serum IgG were added to 293T cells transiently expressing human CD153 and incubated for 20 minutes at 4°C. After washing, 10 μg / ml of rhCD30 / human IgG1 chimeric protein (R&D, 813-CD-100) was added and incubated for 30 minutes at 4°C. After further washing, R-phycoerythrin-labeled anti-human IgG was added and incubated for 20 minutes at 4°C. The decrease in R-phycoerythrin fluorescence intensity was used as an index of inhibitory effect and compared between antibodies. The results are shown in Figures 8 and 9. Figure 8 shows the average value from duplicate wells. Figure 9 shows the results at an antibody concentration of 10 μg / ml.

[0068] From the results of Example 1, 2H7-9 was selected from the three purified antibodies obtained in this Example for further study. The heavy and light chain variable regions of monoclonal antibody 2H7-9 were determined, and each CDR was predicted by the Kabat method. Heavy chain variable region: SEQ ID NO: 15 Light chain variable region: SEQ ID NO: 16 Each predicted CDR is summarized below.

[0069]

[0070] Evaluation of the preventive and therapeutic effects of anti-human CD153 antibody (2H7-9) on EAE. Mice expressing hCD153 and hCD30 (hCD153 / hCD30 knock-in mice) were generated by CRISPR / Cas-mediated genome editing, in which the nucleotide sequences encoding hCD153 and hCD30 were inserted into the endogenous CD153 and CD30 loci, respectively, of mouse zygotes. Female hCD153 / hCD30 knock-in mice were bred with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG) emulsified in complete Freund's adjuvant containing Mycobacterium tuberculosis H37Ra. ３５－５５Mice are immunized subcutaneously (sc) with the 2H7-9 peptide and then intraperitoneally (ip) injected with pertussis toxin to induce EAE. 2H7-9 or PBS (or mouse serum IgG) is administered i.p. before or after the onset of EAE. Clinical symptoms of EAE are evaluated daily. The efficacy of 2H7-9 is evaluated by comparing the incidence and clinical severity of EAE in mice treated with 2H7-9 with those treated with PBS or serum IgG. 2H7-9 suppresses the onset or progression of EAE.

[0071] Reactivity of anti-human CD153 antibodies (2H7-9, 3A2-5, 13H9-13) to canine and feline CD153. Vectors expressing canine and feline CD153 were constructed by inserting DNA sequences encoding canine and feline CD153 proteins into the pIRES2-AcGFP1 vector. 293T cells were transfected with the constructed vectors and cultured at 37°C in a 5% CO2 incubator for 2 days. After culture, the cells were detached from the culture dish using trypsin-EDTA and washed with medium and then with 2% FCS-PBS. Anti-human CD153 antibodies were added to the cells and incubated at 4°C for 20 minutes.

[0072] After washing the cells, R-phycoerythrin-labeled anti-mouse IgG was added and incubated for 20 minutes at 4°C. R-phycoerythrin fluorescence in the vector-transfected cells (AcGFP1-positive fraction) was detected by FACS, and the binding of each anti-human CD153 antibody to canine and feline CD153 was assessed.

[0073] The results are shown in Figure 10. It was confirmed that the anti-human CD153 antibody 2H7-9 reacts with canine and feline CD153, whereas 3A2-5 and 13H9-13 did not react with either canine or feline CD153.

[0074] Preparation of CD153 Chimeric Antigen Receptor (CAR) T Cells An expression vector for the anti-mouse CD153 CAR protein was constructed by inserting into a retroviral vector DNA sequences encoding a single-chain antibody sequence (scFv) in which the heavy chain variable region and light chain variable region of the anti-mouse CD153 antibody clone 17D shown in Reference Examples are linked via a linker peptide, a hinge sequence, a transmembrane domain, a costimulatory molecule intracellular domain, and a CD3ζ intracellular domain.

[0075] The constructed retroviral vector was transfected into platinum-E cells. The culture supernatant containing the viral particles was collected and used to infect CD8-positive T cells isolated from wild-type mouse spleen cells to generate mCD153-CAR-T cells.

[0076] Confirmation of the killing effect of mCD153-CAR-T cells on CD153-expressing cells The mCD153-CAT-T cells obtained in Example 4 were derived from a T cell line (EGPF) that stably expressed mCD153. ＋ ) at an effector:target ratio of 10:1 or 3:1, and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator. After 36 hours of culture, the cells were harvested and SYTOX Red staining agent was added to stain dead cells. The percentage of dead cells in the T cell line was measured by FACS. As a control, non-CAR-transduced CD8-positive T cells isolated from spleen cells of wild-type mice were used.

[0077] The results are shown in Figure 11. Compared to the control group, mCD153-CAR-T cells significantly increased cell death in the mCD153-expressing T cell line, confirming that they exert a killing effect on mCD153-expressing T cells. These results suggest that the increase in SA-T cells in multiple sclerosis can also be suppressed by administering CAR-T cells.

[0078] An expression vector for the anti-human CD153 CAR protein can be constructed in the same manner as in Example 4, using an scFv sequence derived from the anti-human CD153 antibody clone 2H7-9. hCD153-CAR-T cells, which are produced by introducing this vector into human CD8-positive T cells, function according to the same principle as mCD153-CAR-T cells and are capable of killing hCD153-expressing T cells. Thus, hCD153-CAR-T cells demonstrate a killing effect against hCD153-expressing T cells in the same test as in Example 5, except that a T cell line expressing hCD153 is used.

Claims

1. An anti-human CD153 monoclonal antibody, or a binding fragment thereof, that specifically binds to human CD153 and inhibits the intermolecular interaction between human CD153 and CD30.

2. The monoclonal antibody or binding fragment thereof according to claim 1, having the heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-3) shown below.

3. A monoclonal antibody or a binding fragment thereof according to claim 1 or 2, which further binds to feline CD153 or canine CD153.

4. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A pharmaceutical composition for treating a disease involving CD153, comprising the monoclonal antibody or binding fragment thereof according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, wherein the disease in which CD153 is involved is multiple sclerosis.

8. A method for treating or preventing a disease involving CD153 in a subject, comprising administering to a subject in need of such treatment an effective amount of a monoclonal antibody or binding fragment thereof according to any one of claims 1 to 3.

9. The method of claim 8, wherein the subject is a human.

10. The method of claim 9, wherein the subject is a dog or a cat.

11. The method according to any one of claims 8 to 10, wherein the disease in which CD153 is involved is multiple sclerosis.

12. An antibody that contains an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain has the following heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-3): A chimeric antigen receptor, which is a polypeptide comprising a single chain antibody comprising the chimeric antigen receptor.

13. A polynucleotide encoding the chimeric antigen receptor of claim 12.

14. A vector comprising the polynucleotide of claim 13.

15. A CAR-T cell that expresses the chimeric antigen receptor described in claim 12 and has antigen-specific killer activity.

16. A pharmaceutical composition for treating a disease in which CD153 is involved, comprising the CAR-T cells according to claim 15.

17. The pharmaceutical composition according to claim 16, wherein the disease in which CD153 is involved is multiple sclerosis.

18. A method for treating or preventing a disease in which CD153 is involved in a subject, comprising administering an effective amount of the CAR-T cell according to claim 15 to a subject in need of such treatment.

19. The method of claim 18, wherein the subject is a human.

20. The method of claim 18, wherein the subject is a dog or a cat.

21. The method according to any one of claims 18 to 20, wherein the disease in which CD153 is involved is multiple sclerosis.

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