Use of Anti-CD40 antibody in treatment of tumor

Abstract

The present disclosure relates to use of an anti-CD40 antibody in the treatment of a tumor. Specifically, the present disclosure pertains to the field of pharmaceuticals and relates to a method for treating a tumor with an anti-CD40 antibody or an antigen-binding fragment thereof and pharmaceutical use thereof.

Description

Uses of anti-CD40 antibodies in tumor treatment

[0001] This disclosure claims priority to Chinese patent application CN202510347503.4, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure pertains to the pharmaceutical field and relates to methods for treating tumors with CD40 antibodies and their pharmaceutical uses. Background Technology

[0003] CD40 is a cell surface-expressed glycoprotein, a type I intrinsic membrane glycoprotein with a molecular weight of 48 kDa, belonging to the tumor necrosis factor receptor (TNFR) superfamily, and playing an important role in the immune system. It is expressed in various immune cells, such as B cells, dendritic cells, monocytes, and macrophages. When signal transduction occurs via CD40, specialized antigen-presenting cells are activated. The natural ligand of CD40 is named CD154 or CD40L, and is known to be primarily expressed in mature T lymphocytes. CD40L-mediated signal transduction triggers several cellular biological events, including immune cell activation, proliferation, and the production of cytokines and chemokines. CD40 signaling is crucial for T cell-dependent immune responses, especially in a tumor environment. CD40-stimulated dendritic cells can activate tumor-specific effector T cells, which have the potential to eradicate tumor cells.

[0004] CD40 expression occurs in many normal cells and tumor cells, including B lymphocytes. For example, melanoma is a type of tumor that expresses CD40, and 30% to 70% of solid tumors also express CD40. It is known that CD40 activation can effectively trigger anti-tumor responses (Tong et al., Cancer Gene Therapy, 2003, 10:1-13), including immune activation of tumor-specific T cell responses, direct apoptosis in CD40-positive tumors, and humoral responses to ADCC induced by stimulation. Furthermore, observed tumor eradication is strongly correlated with the emergence of tumor-specific cytotoxic T lymphocytes. However, it should not be overlooked that systemic administration of CD40 antibodies is associated with side effects such as shock syndrome and cytokine release syndrome (van Mierlo et al., Proc. Natl. Acad. Sci. USA, 2002, 99:5561-5566).

[0005] Therefore, developing safer and more effective immunotherapy regimens for tumors using CD40 antibodies is an important issue that needs to be addressed in the field of targeted immunotherapy for tumors. Summary of the Invention

[0006] This disclosure provides a method for treating tumors with anti-CD40 antibodies and their pharmaceutical applications.

[0007] In some embodiments, this disclosure provides the use of anti-CD40 antibodies or antigen-binding fragments thereof in the preparation of medicaments for treating tumors.

[0008] In some embodiments, this disclosure provides an anti-CD40 antibody or an antigen-binding fragment thereof in a medicament for treating tumors.

[0009] In some embodiments, this disclosure provides a method of treating tumors, including administering a therapeutically effective amount of an anti-CD40 antibody to a subject in need.

[0010] In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a malignant solid tumor.

[0011] In this disclosure, "malignant solid tumor" refers to a tumor that is carcinogenic. Carcinogenic tumors can invade surrounding tissues within the body, and as these tumors grow, some cancer cells may migrate to other parts of the body to form other "secondary" tumors, also known as metastases.

[0012] In this disclosure, systemic anti-tumor treatment regimens are well known to those skilled in the art, such as those disclosed in regulatory-approved treatment guidelines. Examples include, but are not limited to, detailed and up-to-date information on standard treatment regimens for various cancers provided in NCCN and CSCO guidelines.

[0013] In some implementations, the tumor is bladder cancer.

[0014] In some implementations, the tumor is non-muscle-invasive bladder cancer.

[0015] In some implementations, the tumor is intermediate- or high-risk non-muscle-invasive bladder cancer.

[0016] In some implementations, the non-muscle-invasive bladder cancer is intermediate-risk non-muscle-invasive bladder cancer that has failed BCG or chemotherapy treatment or is intolerant to BCG or chemotherapy.

[0017] In some implementations, the non-muscle-invasive bladder cancer is high-risk non-muscle-invasive bladder cancer that is unresponsive to BCG treatment, unsuitable for BCG bladder instillation therapy, or unsuitable for or unwilling to undergo radical cystectomy.

[0018] Anti-CD40 antibody

[0019] In some embodiments, the anti-CD40 antibody or antigen-binding fragment has LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively, and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0020] Table 1. CDR Sequences

[0021] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% or at least 90% identity with it, and the VL comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% or at least 90% identity with it.

[0022] In some implementations, the anti-CD40 antibody or antigen-binding fragment may be selected from murine antibodies, chimeric antibodies, humanized antibodies, or their antigen-binding fragments.

[0023] In some specific embodiments, the anti-CD40 antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In some embodiments, the light chain and heavy chain FR region sequences on the variable regions of the humanized CD40 antibody are derived from human germline light and heavy chains or their mutant sequences, respectively.

[0024] In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% or at least 90% identity with it, and the VL comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% or at least 90% identity with it.

[0025] >hu9E5-H1c:

[0026] >hu9E5-L1a:

[0027] In some embodiments, the humanized CD40 antibody heavy chain sequence is the sequence shown in SEQ ID NO:9 or a variant thereof, wherein the variant preferably has 0-10 amino acid changes in the variable region of the heavy chain, more preferably mutations at amino acid sites 6 and 8, and the mutated amino acids are preferably I, A or L; the humanized antibody light chain sequence is the sequence shown in SEQ ID NO:10 or a variant thereof, wherein the variant preferably has 0-10 amino acid changes in the variable region of the light chain, more preferably mutations at amino acid sites 2 and 3, and the mutated amino acids are preferably I, V or L.

[0028] In some embodiments, the anti-CD40 antibody further comprises an Fc region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof; in some embodiments, the heavy chain variable region of the anti-CD40 antibody comprises an Fc region of human IgG1, IgG2 or IgG4 or a variant thereof; in some embodiments, the heavy chain variable region of the CD40 antibody comprises an Fc region of human IgG1 or IgG2.

[0029] In some embodiments, the anti-CD40 antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in or having at least 80% or at least 90% identity with SEQ ID NO: 13, and the light chain comprising an amino acid sequence as shown in or having at least 80% or at least 90% identity with SEQ ID NO: 14.

[0030] >hu9E5 HC:

[0031] >hu9E5 LC:

[0032] In the context of this disclosure, "at least 80%" encompasses 80% and above, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any numerical range between any two.

[0033] In some embodiments, the heavy chain of the anti-CD40 antibody contains an amino acid mutation relative to SEQ ID NO: 13, wherein the amino acid mutation is selected from S267E, N325S, and L328F (determined by natural counting). In some embodiments, the last amino acid K in the anti-CD40 antibody heavy chain may be replaced with A.

[0034] In some embodiments, the heavy chain of the anti-CD40 antibody comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% or at least 90% identity with it, and the light chain of the anti-CD40 antibody comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% or at least 90% identity with it.

[0035] >hu9E5-SELFNS HC:

[0036] >hu9E5-SELFNS LC

[0037] In some embodiments, the preparation of the above-mentioned anti-CD40 antibody or its antigen-binding fragment can refer to WO2020108611A1 and WO2020108621A1. This disclosure incorporates, by reference, the relevant contents of antibody sequences, preparation methods, and compositions in WO2020108611A1 and WO2020108621A1.

[0038] In some embodiments, the dosage of the anti-CD40 antibody or its antigen-binding fragment is 1 mg to 5000 mg, for example 1 mg to 4500 mg, 1 mg to 4000 mg, 1 mg to 3500 mg, 1 mg to 3000 mg, 1 mg to 2500 mg, 1 mg to 2000 mg, 1 mg to 1500 mg, 2 mg to 3500 mg, 2 mg to 3000 mg, 2 mg to 2500 mg, 2 mg to 2000 mg, 2 mg to 1500 mg, 3 mg to 3500 mg, 3 mg to 3000 mg, 3 mg to 2500 mg, 3 mg to 2000 mg, 3 mg to 1500 mg, or 4 mg to 1500 mg, 4 mg to 1800 mg, 5 mg to 1800 mg, 100 mg to 1000 mg, 100 mg to 800 mg; and any range of values ​​between any two.

[0039] In some embodiments, the dosage of the anti-CD40 antibody or its antigen-binding fragment is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 8 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 26 mg, about 28 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, 225 mg, about 250 mg, about 300 mg, or about 375 mg. Approximately 400mg, approximately 425mg, approximately 450mg, approximately 475mg, approximately 500mg, approximately 550mg, approximately 600mg, 650mg, approximately 700mg, approximately 750mg, approximately 800mg, approximately 850mg, approximately 900mg, approximately 950mg, approximately 1000mg, approximately 1050mg, approximately 1100mg, approximately 1150mg, approximately 1200mg, approximately 1250mg, approximately 1300mg, approximately 1350mg, approximately 1400mg, approximately 1450mg, approximately 1500mg, 1550mg, approximately 1600mg g, approximately 1650mg, approximately 1700mg, approximately 1750mg, approximately 1800mg, approximately 1850mg, approximately 1900mg, approximately 1950mg, 2000mg, approximately 2050mg, approximately 2100mg, approximately 2150mg, approximately 2200mg, approximately 2250mg, approximately 2300mg, approximately 2350mg, approximately 2400mg, 2450mg, approximately 2500mg, approximately 2550mg, approximately 2600mg, approximately 2650mg, approximately 2700mg, approximately 2750mg, approximately 2800mg, approximately 2850mg g, 2900mg, about 2950mg, about 3000mg, about 3050mg, about 3100mg, about 3150mg, about 3200mg, about 3250mg, about 3300mg, about 3350mg, about 3400mg, 3450mg, about 3500mg, about 3550mg, about 3600mg, about 3650mg, about 3700mg, about 3750mg, about 3800mg, about 3850mg, 3900mg, about 3950mg, about 4000mg, about 4500mg, about 4800mg.

[0040] In some implementations, the dose of the anti-CD40 antibody or its antigen-binding fragment is about 300 mg or about 600 mg.

[0041] In some embodiments, the frequency of administration of the anti-CD40 antibody or its antigen-binding fragment is selected from at least once every 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, or 10 weeks. In some specific embodiments, the frequency of administration of the anti-CD40 antibody or its antigen-binding fragment is selected from at least once every 1 week, 2 weeks, 3 weeks, 4 weeks, or 6 weeks. In some embodiments, the frequency of administration of the anti-CD40 antibody or its antigen-binding fragment is once every 1 week, 2 weeks, 3 weeks, 4 weeks, or 6 weeks.

[0042] In some embodiments, the anti-CD40 antibody is administered via bladder instillation. In some embodiments, the administration regimen of the anti-CD40 antibody or its antigen-binding fragment includes an induction instillation period and a maintenance instillation period. In some embodiments, during the induction instillation period, the anti-CD40 antibody or its antigen-binding fragment is instilled once weekly; in some embodiments, during the induction instillation period, the anti-CD40 antibody or its antigen-binding fragment is instilled once weekly; for 1-12 consecutive instillations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 times). In some embodiments, during the maintenance instillation period, the anti-CD40 antibody or its antigen-binding fragment is instilled once weekly, and three times monthly. In some embodiments, during the maintenance perfusion period, the anti-CD40 antibody or its antigen-binding fragment is perfused once a week, and the anti-CD40 antibody or its antigen-binding fragment is perfused three times a month; for a total of 9-30 perfusions (e.g., 9, 12, 15, 18, 21, 24, 27, or 30 perfusions). In some embodiments, the maintenance perfusion period is 3, 6, 12, 18, or 24 months after the induction perfusion period.

[0043] Administering anti-CD40 antibodies to subjects using the dosing regimen provided in this disclosure can significantly improve the objective response rate (ORR), prolong progression-free survival (PFS), and / or overall survival (OS) in cancer patients, providing a safe and effective treatment option for cancer patients and bringing clinical benefits superior to existing treatments.

[0044] the term

[0045] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0046] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as having an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.”

[0047] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0048] The terms "about" and "approximately" mean that a numerical value is within the acceptable error range of a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially includes" may mean a range of up to 10%, and in this disclosure, each instance of the term "about" preceding a number or range of values ​​also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable error range of that specific value.

[0049] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0050] In some implementations, this disclosure defines the following:

[0051] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0052] As used herein, the term "antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (CDRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0053] The determination or definition of a contact ligand (CDR) can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0054] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16; 9:2278), etc.

[0055] The terms “antigen-binding fragment” or “functional fragment” or “antigen-binding portion” refer to one or more fragments of a complete antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Exemplary examples of binding fragments encompassed in the term “antigen-binding fragment” include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by disulfide bridges on their hinge regions; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of the antibody; (v) dsFv, a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; (vi) scFv; and (vii) bispecific, bispecific, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab.

[0056] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -8 M, for example, approximately less than 10 -9 M, 10 -10 M, 10 -11 M or lower affinity (KD) binding.

[0057] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies disclosed herein have a KD value less than approximately 10⁻⁷ M, for example, less than approximately 10⁻⁷ M. -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (KD) is combined with IL-5, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology.

[0058] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same bases or amino acid monomer subunits; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of homology. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm frequently used in sequence analysis: BLAST Algorithms: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other common BLAST algorithms, such as those provided by NCBI BLAST, are also well-known to those skilled in the art.

[0059] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0060] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing methods commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0061] An "effective amount" includes the amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0062] The terms “subject” and “patient” refer to mammals, especially primates, and particularly humans.

[0063] The term "bladder cancer" is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) based on whether the tumor infiltrates the bladder muscle layer. NMIBC includes CIS stage (carcinoma in situ), Ta stage, and T1 stage.

[0064] In non-muscle-invasive bladder cancer, stage "Ta" is non-invasive papillary carcinoma, which has not invaded the connective tissue or deep muscle layer of the bladder wall. Stage "Tis" is flat carcinoma, also known as carcinoma in situ (CIS), which has not invaded the connective tissue or deep muscle layer of the bladder wall. In stage "T1", cancer cells have grown into the connective tissue layer but have not yet reached the muscle layer of the bladder wall. Attached Figure Description

[0065] Figure 1 shows the modeling and drug administration of an orthotopic bladder cancer pharmacodynamic model in hCD40 humanized mouse MB49-luc cells.

[0066] Figure 2 shows the survival curves of mice in the G1 and G2 treatment groups. Detailed Implementation

[0067] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor Laboratory's *Antibody Technology Laboratory Manual* and *Molecular Cloning Manual*; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available conventional reagents. Example 1. Phase I Clinical Study of Anti-CD40 Antibody Bladder Instillation for Intermediate-High Risk Non-Muscle-Invasive Bladder Cancer (NMIBC)

[0068] 1.1 Test Drug

[0069] Anti-CD40 antibody, its heavy chain sequence is shown in SEQ ID NO:9, and its light chain sequence is shown in SEQ ID NO:10. Dosage form: injection, strength: 1ml:25mg. Route of administration: bladder instillation.

[0070] Administration method: Before instillation, empty the urine through a urinary catheter, dilute the anti-CD40 antibody to 50 mL of normal saline, and instill it into the bladder through the urinary catheter. The instillation solution is kept in the bladder for 2 hours ± 10 minutes.

[0071] 1.2 Enrolled Subjects

[0072] (1) Age ≥ 18 years old, gender not limited;

[0073] (2) Previous pathological tissue biopsy diagnosis of intermediate or high-risk NMIBC (“intermediate and high risk” are defined in the table below);

[0074] Table 2. Risk stratification of non-muscle-invasive bladder cancer (CSCO 2023)

[0075] (3) Eastern Cooperative Oncology Group (ECOG) performance status score: 0 or 1;

[0076] (4) Expected survival time ≥ 2 years;

[0077] (5) No resectable lesions were found during cystoscopy within 6 weeks prior to the first dose, or the only remaining lesion was CIS; for T1 subjects, if the pathological examination of the resected tissue after the last transurethral resection of bladder tumor (TURBT) did not show the muscle layer, TURBT must be performed again and the pathological examination must confirm that the muscle layer tissue is included.

[0078] (6) For intermediate-risk NMIBC, those who have failed BCG / chemotherapy or are intolerant to BCG / chemotherapy;

[0079] For high-risk NMIBC, those who do not respond to BCG treatment, those who are not suitable for BCG bladder instillation therapy, and those who are not suitable for or unwilling to undergo radical cystectomy;

[0080] Not suitable for BCG bladder instillation definition:

[0081] - Past medical history or auxiliary examinations suggest a history of active tuberculosis within the past year;

[0082] - Patients who have previously discontinued treatment due to severe adverse reactions to BCG bladder instillation;

[0083] - Insufficient BCG production capacity, no BCG available at local treatment centers, and intolerance to chemotherapy drugs or progression after treatment with ≥1 chemotherapy drug;

[0084] -BCG-related allergic reactions;

[0085] -Other situations deemed unsuitable for BCG bladder instillation therapy by researchers;

[0086] Definition of unsuitable for radical cystectomy:

[0087] -The patient is unable to tolerate major surgery due to weak physical condition or underlying diseases;

[0088] - A history of abdominal or pelvic surgery / radiotherapy has caused adhesions of the abdominal and pelvic organs, making radical cystectomy impossible.

[0089] - Other cases where researchers have assessed that the patient is not suitable for radical cystectomy.

[0090] (7) Subjects in the efficacy expansion phase need to meet one of the following conditions:

[0091] - Cohort 1: No prior BCG treatment;

[0092] - Cohort 2: CIS patients who did not respond to previous BCG treatment* (with or without Ta / T1);

[0093] - Cohort 3: Ta or T1 patients (without CIS) who have not responded to previous BCG treatment*;

[0094] - Cohort 4: Intermediate-risk NMIBC who have failed previous BCG or chemotherapy.

[0095] (*BCG non-response is defined as meeting any of the following criteria: ①. Persistent / recurrent CIS or combined Ta / T1 within 12 months of adequate BCG treatment; ②. Recurrence of high-grade Ta / T1 within 6 months of adequate BCG treatment; ③. High-grade T1 is present on the first assessment after BCG induction perfusion. Adequate BCG treatment as described above includes: completion of 5 out of 6 initial induction perfusions and 2 out of 3 maintenance perfusions, or completion of 5 out of 6 initial induction perfusions and 2 out of 6 re-induction perfusions.)

[0096] (8) As defined by the following laboratory test results, there is a sufficient level of organ function (no blood transfusions or blood products within 14 days prior to the first dose, and no correction with G-CSF or other hematopoietic stimulating factors):

[0097] a) Complete blood count: Absolute neutrophil count (ANC) ≥ 1.5 × 10⁻⁶ 9 / L; Platelet count (PLT) ≥100×10 9 / L; Hemoglobin (Hb) ≥90g / L;

[0098] b) Liver function tests: Total bilirubin (TBIL) ≤ 1.5 × upper limit of normal (ULN); serum transaminase ALT and / or AST ≤ 2.5 × ULN; albumin > 3 g / dL;

[0099] c) Kidney function tests: serum creatinine (Cr) ≤ 1.5 × ULN or creatinine clearance ≥ 60 ml / min (calculated according to the Cockcroft-Gault formula);

[0100] d) Electrocardiogram examination: QTcF ≤ 450ms for males and QTcF ≤ 470ms for females;

[0101] e) Echocardiography: Left ventricular ejection fraction (LVEF) ≥ 50%;

[0102] f) Coagulation function test: APTT≤1.5×ULN, and INR or PT≤1.5×ULN.

[0103] 1.3 Research Plan

[0104] Phase 1: Dosage escalation phase

[0105] Four dose groups were pre-set for dose escalation: 25 mg, 100 mg, 300 mg, and 600 mg. The dosing cycle included a 6-week induction perfusion period (once a week for 6 consecutive weeks) and a 15-week maintenance perfusion period (once a week for 3 weeks before the first induction perfusion at 3, 6, 12, 18, and 24 months).

[0106] For high-risk NMIBC patients who are unresponsive to BCG, if a subject is found to have recurrent or persistent CIS or high-grade Ta by cystoscopy before the first bladder instillation 3 months after the first induction instillation; for high-risk NMIBC patients who are untreated by BCG, if a subject is found to have recurrent or persistent CIS or high-grade Ta or T1 (without pathological progression) by cystoscopy before the first bladder instillation 3 months after the first induction instillation, then 3 additional instillations (once a week) should be added after completing the routine 3 instillations this month as re-induction instillations.

[0107] Phase Two: Therapeutic Effect Expansion Phase

[0108] This phase will include the following four categories of participants:

[0109] 1) Cohort 1: No prior BCG treatment

[0110] 2) Cohort 2: Carcinoma in situ (CIS) that has not responded to previous BCG (with or without Ta or T1);

[0111] 3) Queue 3: Ta or T1 who have previously not responded to BCG (without CIS);

[0112] 4) Cohort 4: Intermediate-risk NMIBC with previous BCG or chemotherapy failure.

[0113] The specific dosing cycle and dosing schedule (including re-induction perfusion) are the same as in the first phase.

[0114] 1.4 Results Evaluation

[0115] (1) Evaluation of efficacy: Cystoscopy (biopsy if necessary) and urine cytology will be used to evaluate efficacy. The study will assess the CR rate and DOR in the CIS population and the total population, the DFS rate in the non-CIS population and the total population, and the DFS rate in the total population at 6, 12 and 24 months.

[0116] (2) Safety assessment: Adverse events, serious adverse events (SAEs), vital signs and physical examinations, 12-lead ECGs, and laboratory tests were collected to assess safety. The severity of AEs was graded using the NCI-CTCAE v5.0 standard.

[0117] Example 2. Efficacy evaluation of anti-CD40 antibody in combination with hCD40 humanized mouse MB49 orthotopic bladder cancer

[0118] Efficacy evaluation of a humanized hCD40 mouse MB49-luc cell orthotopic bladder cancer model: On day 3 (6-8 weeks), female humanized hCD40 mice were anesthetized by intraperitoneal injection of aphthylamine. After being positioned appropriately, a bladder catheter of appropriate size was inserted. The bladder was first rinsed with PBS, then instilled with an appropriate concentration of hydrochloric acid for 30 seconds, followed by PBS instillation until the outflow fluid became neutral. A certain amount of MB49-luc cell suspension was then instilled into the mouse bladder and kept full for 1 hour, during which time the mice were kept under anesthesia. Starting on day 0, the model mice were treated with bladder instillation twice a week. The grouping and treatment methods are shown in Table 1.

[0119] Because the anti-CD40 antibody in Example 1 contains human IgG1, which has poor affinity for mouse Fc receptors, mouse IgG1 was used instead of molecular anti-CD40 antibody-mIgG1. The dosage was calculated based on the highest permissible clinical concentration of anti-CD40 antibody (25 mg / mL, 50 μL / mouse). However, due to the anti-CD40 antibody-mIgG1 concentration of 16.71 mg / mL, the dosage was adjusted to 80 μL to achieve a dose of 1.25 mg / dose.

[0120] Heavy chain of anti-CD40 antibody-mIgG1

[0121] The light chain of anti-CD40 antibody-mIgG1

[0122] Table 3. Dosage

[0123] Table 4. Median survival

[0124] Example 3. Phase Ib / II Clinical Study of Anti-CD40 Antibody Monotherapy for Unresectable Intermediate-High Risk Non-Muscle-Invasive Bladder Cancer (NMIBC)

[0125] 1. Test drug

[0126] Anti-CD40 antibody, its heavy chain sequence is shown in SEQ ID NO:9, and its light chain sequence is shown in SEQ ID NO:10. Dosage form: injection, strength: 1ml:25mg. Route of administration: bladder instillation.

[0127] 2. Enrolled subjects

[0128] 1) Male or female, aged ≥18 years;

[0129] 2) Pathological tissue biopsy diagnosis of intermediate or high-risk NMIBC;

[0130] 3) TURBt cannot be completely removed (at least two senior urologists assess that the lesion is extensive and cannot be completely removed by electrocautery);

[0131] 4) Unsuitable for or unwilling to undergo radical cystectomy (patients who cannot tolerate major surgery due to weak physical condition or underlying diseases; those with a history of abdominal or pelvic surgery / radiotherapy causing adhesions of abdominal and pelvic organs that prevent radical cystectomy from being performed; other situations deemed unsuitable for radical cystectomy by researchers).

[0132] 5) The most recent TURBt was conducted within the last 6 weeks;

[0133] 6) The subject was willing to provide cystoscopic biopsy specimens for review;

[0134] 7) Physical condition assessment (ECOG score): 0-2 points;

[0135] 8) Expected survival ≥ 2 years;

[0136] 9) As defined by the following laboratory test results, there is sufficient organ function (no blood transfusions or blood products within 14 days prior to the first dose, and no correction with G-CSF or other hematopoietic stimulating factors):

[0137] a) Complete blood count: Absolute neutrophil count (ANC) ≥ 1.5 × 10⁹ / L; Platelet count (PLT)

[0138] ≥100×10⁹ / L; Hemoglobin (Hb) ≥90g / L;

[0139] b) Liver function tests: Total bilirubin (TBIL) ≤ 1.5 × upper limit of normal (ULN); serum transaminase ALT and / or AST ≤ 2.5 × ULN; albumin > 3 g / dL;

[0140] c) Kidney function tests: serum creatinine (Cr) ≤ 1.5 × ULN or creatinine clearance ≥ 60 ml / min (calculated according to the Cockcroft-Gault formula); urinalysis showing ≥ ++ urine protein requires confirmation that 24-hour urine protein ≤ 1.0 g;

[0141] d) Electrocardiogram examination: QTcF ≤ 450ms for males and QTcF ≤ 470ms for females;

[0142] e) Echocardiography: Left ventricular ejection fraction (LVEF) ≥ 50%;

[0143] f) Coagulation function test: APTT≤1.5×ULN, and INR or PT≤1.5×ULN.

[0144] 10) Female participants should be surgically sterilized or postmenopausal, or agree to use at least one medically approved method of contraception (such as an intrauterine device, birth control pill, or condom) during and for 6 months after the study treatment period. A negative blood pregnancy test must be taken within 7 days prior to study enrollment; false positive results may be ruled out by the investigator before enrollment. Male participants should agree to use at least one medically approved method of contraception during and for 6 months after the study treatment period.

[0145] 11) Participants voluntarily joined this study, signed informed consent forms, demonstrated good compliance, and cooperated with follow-up.

[0146] 3. Administration method

[0147] Anti-CD40 antibody monotherapy group: administration via bladder instillation; the first 6 weeks are the induction instillation period, with instillation once a week for a total of 6 weeks; after the completion of induction instillation (3 months after the first dose), efficacy is evaluated, and patients with complete remission (CR) enter the maintenance instillation period, with instillation once a week (i.e., 3 times a month for a total of 15 weeks) for the first 3, 6, 12, 18 and 24 months after the first induction instillation; the dose is 600 mg, and adjustments to a lower dose that has been validated during the dose escalation phase are allowed.

[0148] 4. Results Evaluation

[0149] 4.1 Validity Analysis:

[0150] Data are from the NMIBC-IIT study (cutoff date: February 23, 2026). A total of 11 patients with unresectable intermediate- to high-risk non-muscle-invasive bladder cancer were enrolled in the 300mg and 600mg anti-CD40 antibody groups. Four patients were enrolled in the 300mg group, all of whom completed at least one efficacy evaluation. Seven patients were enrolled in the 600mg group, including three in the anti-CD40 antibody monotherapy group. All patients completed at least one efficacy evaluation.

[0151] Table 5. Validity Results

[0152] Note: CR, complete remission; PR, partial remission; PD, disease progression.

[0153] Table 1 lists the preliminary treatment outcomes of patients with unresectable intermediate- to high-risk non-muscle-invasive bladder cancer treated with the 600 mg dose of anti-CD40 antibody. In the 600 mg anti-CD40 antibody monotherapy group, three patients were evaluable for efficacy, with a 3-month complete response (CR) rate of 66.7% (2 / 3).

[0154] 4.2 Security Analysis:

[0155] As of February 23, 2026, the most common treatment-related adverse events (TRAEs) of anti-CD40 antibodies were hematuria (18.2%, 2 / 11) and urinary tract infection (9.1%, 1 / 11). The incidence of CTCAE ≥ 3 TEAEs was 9.1% (1 / 11).

[0156] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. The use of anti-CD40 antibody or its antigen-binding fragment in the preparation of drugs for treating tumors; wherein, The CD40 antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; the tumor is non-muscle-invasive bladder cancer.

2. The use according to claim 1, wherein, The dosage of the anti-CD40 antibody or its antigen-binding fragment is 1-5000 mg, preferably about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, or about 800 mg. Preferably, the frequency of administration of the anti-CD40 antibody or its antigen-binding fragment is selected from at least once every 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks or 10 weeks, and preferably once every 1 week, 2 weeks, 3 weeks, 4 weeks or 6 weeks.

3. The use according to claim 1 or 2, wherein, The anti-CD40 antibody or its antigen-binding fragment is administered via bladder instillation; preferably, the administration regimen of the anti-CD40 antibody or its antigen-binding fragment includes an induction instillation period and a maintenance instillation period; wherein, during the induction instillation period, the anti-CD40 antibody or its antigen-binding fragment is instilled once a week; during the maintenance instillation period, the anti-CD40 antibody or its antigen-binding fragment is instilled once a week, and the anti-CD40 antibody or its antigen-binding fragment is instilled three times a month.

4. The use according to any one of claims 1-3, wherein, The anti-CD40 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment; Preferably, the light chain and heavy chain FR region sequences on the variable regions of the humanized antibody light chain and heavy chain are derived from human germline light chain and heavy chain or their mutant sequences, respectively.

5. The use according to any one of claims 1-4, wherein, The anti-CD40 antibody or its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL); The VH contains an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% or at least 90% sequence identity with it, and the VL contains an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% or at least 90% sequence identity with it; Alternatively, the VH may contain an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% or at least 90% sequence identity with it, and the VL may contain an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% or at least 90% sequence identity with it.

6. The use according to any one of claims 1-5, further comprising an Fc region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

7. The use according to any one of claims 1-6, wherein the anti-CD40 antibody comprises a heavy chain and a light chain; wherein, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% or at least 90% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% or at least 90% sequence identity with it; or, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:13 or having at least 80% or at least 90% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:14 or having at least 80% or at least 90% sequence identity with it.

8. The use according to any one of claims 1-7, wherein, The non-muscle-invasive bladder cancer mentioned is intermediate-risk or high-risk non-muscle-invasive bladder cancer; Preferred options are: (1) The non-muscle-invasive bladder cancer referred to is intermediate-risk non-muscle-invasive bladder cancer that has failed BCG or chemotherapy treatment or is intolerant to BCG or chemotherapy; or (2) The non-muscle-invasive bladder cancer mentioned refers to high-risk non-muscle-invasive bladder cancer that does not respond to BCG treatment, is not suitable for BCG bladder instillation therapy, or is not suitable for or unwilling to undergo radical cystectomy.

9. A method of treating a tumor, comprising administering to a subject in need a therapeutically effective amount of an anti-CD40 antibody or an antigen-binding fragment thereof; wherein, The CD40 antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; the tumor is non-muscle-invasive bladder cancer; Preferably, the anti-CD40 antibody or its antigen-binding fragment is as defined in any one of claims 1-7.

10. The method according to claim 9, wherein, The non-muscle-invasive bladder cancer mentioned is intermediate-risk or high-risk non-muscle-invasive bladder cancer; Preferred options are: (1) The non-muscle-invasive bladder cancer referred to is intermediate-risk non-muscle-invasive bladder cancer that has failed BCG or chemotherapy treatment or is intolerant to BCG or chemotherapy; or (2) The non-muscle-invasive bladder cancer mentioned refers to high-risk non-muscle-invasive bladder cancer that does not respond to BCG treatment, is not suitable for BCG bladder instillation therapy, or is not suitable for or unwilling to undergo radical cystectomy.

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