Use of bleomycin acting as immunoenhancer

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to the use of bleomycin or a bleomycin analog in the preparation of an immunoenhancer. Further provided in the present invention are a pharmaceutical combination and the use thereof in the preparation of a drug for treating and / or preventing cancers, wherein the pharmaceutical combination contains bleomycin, a bleomycin analog or a combination thereof, and an anti-tumor immunotherapeutic agent.

Description

Use of bleomycin as an immune enhancer

[0001] This application claims priority to Chinese Patent Application No. 202410658244.2, filed May 24, 2024, entitled “Use of bleomycin as an immune enhancer,” the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and specifically relates to the use of bleomycin or a bleomycin analog in the preparation of an immune enhancer. The present application also provides a pharmaceutical combination and the use thereof in the preparation of a medicament for treating and / or preventing cancer, wherein the pharmaceutical combination comprises bleomycin, a bleomycin analog, or a combination thereof, and an anti-tumor immunotherapy agent. BACKGROUND

[0003] Major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules involved in antigen presentation and T cell activation play a key role in anti-tumor and anti-infective immune responses.

[0004] MHC class I molecules are widely distributed on the surface of almost all cells, and their main function is to present endogenous antigens, especially playing a major role in tumor antigen presentation. MHC class II molecules are mainly distributed on the surface of professional antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells, and their main function is to present exogenous antigens, such as viral or bacterial antigens. However, MHC class II molecules can also be expressed by cancer cells, and tumor cells that lack expression of some MHC class I molecules will retain expression of MHC class II molecules. For example, expression of MHC class II molecules and tumor cell-associated pathway components has been found in various human tumor cells, such as melanoma, classical Hodgkin lymphoma, etc.

[0005] In the immune response, T cell receptors (TCRs) on CD4+ T cells and CD8+ T cells recognize MHC-II and MHC-I presented antigen peptides, respectively, leading to activation of T cells into effector T cells. It is generally accepted that direct cytotoxicity against target cells relies on CD8+ T cell effector functions, whereas CD4+ T cell effector functions are considered to be centered on cytokine production. However, these functions are plastic. Preclinical and clinical studies have identified cytotoxic CD4+ T cells that have cytotoxic programs and can directly kill target cells. These cytotoxic CD4+ T cells not only express key molecules associated with cytolytic granules such as granzymes (GZMs) and perforin (PRF1), but also have direct cytotoxicity, which is the basis of pathogenic and protective immunity, including in cancer (see Oh DY and Fong L. Cytotoxic CD4+ T cells in cancer: Expanding the immune effector toolbox. Immunity. 2021 Dec 14;54(12):2701-2711).

[0006] Human MHC molecules are commonly referred to as human leukocyte antigens (HLAs). It is now well established that HLA molecules play a critical role in viral and tumor immune evasion. For example, in multiple cancer types, including head and neck squamous cell carcinoma, breast cancer, colon cancer, ovarian cancer, melanoma, Hodgkin lymphoma, non-small cell lung cancer, and bladder cancer, downregulation of MHC-I is associated with disease progression and poor prognosis. Moreover, previous studies have shown that reduced MHC-I expression is closely related to resistance to immune checkpoint therapy (ICT). And recent studies have shown that tumor cells that simultaneously express MHC-I and MHC-II restricted tumor neoantigens are best responders to immune checkpoint therapy (ICT).

[0007] Immuno-therapy has achieved remarkable clinical efficacy in multiple tumor types. However, despite these impressive advances, there is still considerable variation in the response rate of immunotherapy across different tumor types. And after immunotherapy, tumor size is not long-term controlled in all responding patients. Therefore, the need to discover new therapies and combination strategies to improve the efficacy of cancer immunotherapy has not been met. Malignant cells have a variety of mechanisms to evade immune surveillance, including inducing immune checkpoint expression, establishing an immunosuppressive microenvironment, lower neoantigen load, reduced antigen presentation, and HLA heterozygous loss. Insufficient major histocompatibility complex class I (MHC-I) molecules on the surface of tumor cells is a major obstacle to the success of T cell-mediated immunotherapy.

[0008] Therefore, there is a need for drugs that can increase the immune response and effective strategies for treating cancer or infection using such drugs. SUMMARY

[0009] In one aspect, the present application provides a pharmaceutical combination comprising bleomycin, a bleomycin analog, or a combination thereof, and an immunotherapeutic agent involved in MHC-I and / or MHC-II dependent immune response.

[0010] In one embodiment, the bleomycin is selected from the group consisting of BLM A2, BLM B2, BLM A5, and combinations thereof. In a preferred embodiment, the bleomycin is a mixture of BLM A2 and BLM B2.

[0011] In one embodiment, the bleomycin analog is selected from the group consisting of peliomycin, rachelmycin, liblomycin, zorbamycin, and combinations thereof.

[0012] In one embodiment, the immunotherapeutic agent targets an antigen peptide-MHC-I complex (pMHC-I) and / or an antigen peptide-MHC-II complex (pMHC-II), the antigen peptide being selected from the group consisting of a tumor antigen peptide, a viral antigen peptide, a bacterial antigen peptide, a fungal antigen peptide, and a parasitic antigen peptide. In one embodiment, the antigen peptide is a tumor antigen peptide.

[0013] In one embodiment, the immunotherapeutic agent is an anti-tumor immunotherapeutic agent.

[0014] In one embodiment, the anti-tumor immunotherapeutic agent targets an antigen peptide-MHC-I complex (pMHC-I) and / or an antigen peptide-MHC-II complex (pMHC-II), wherein the antigen peptide is a tumor antigen peptide.

[0015] In one embodiment, the tumor antigen peptide comprises an MHC-I restricted epitope selected from the group consisting of KRAS, MAGE1, gp100, hTERT, NY-ESO-1, hCGβ, Her2 / Neu, Melan-A / MART-1, TARP, p53, p68, MIF, Proteinase 3 (PR1), WT1, HA-1, PRAME, CEA, MAGE-A3, and MAGE-A4. In one embodiment, the tumor antigen peptide comprises an MHC-I restricted epitope selected from the group consisting of KRAS, gp100, and p53.

[0016] In one embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of an antibody, an antibody-drug conjugate, an immune cell, and combinations thereof.

[0017] In an embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), T cell receptor engineered T cells (TCR-T), and combinations thereof.

[0018] In an embodiment, the anti-tumor immunotherapeutic agent comprises a T cell receptor (TCR)-like antibody.

[0019] In an embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of: a TCR-like antibody, a T cell engager, an immunotoxin, a chimeric antigen receptor T cell (CAR-T), and combinations thereof.

[0020] In an embodiment, the T cell engager is a T cell engaging bispecific antibody. In an embodiment, the T cell engaging bispecific antibody is selected from the group consisting of antibody H2-scDb, antibody V2-scDb, and tebentafusp.

[0021] In an embodiment, the immunotherapeutic agent is selected from the group consisting of an immune checkpoint inhibitor, a vaccine, and combinations thereof. In an embodiment, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, and combinations thereof. In an embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody. In an embodiment, the vaccine is a tumor neoantigen-based vaccine.

[0022] In another aspect, the present application provides a pharmaceutical composition comprising the pharmaceutical combination of the present application and a pharmaceutically acceptable carrier.

[0023] The present application also provides a kit comprising the pharmaceutical combination or the pharmaceutical composition of the present application.

[0024] In yet another aspect, the present application also provides the use of the pharmaceutical combination, the pharmaceutical composition or the kit of the present application in the manufacture of a medicament for the treatment or prevention of a cancer or an infectious disease.

[0025] In an embodiment, the cancer is a cancer in which cancer cells express low levels of MHC-I and / or MHC-II.

[0026] In an embodiment, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, bone cancer, prostate cancer, lung cancer, adrenal cancer, bile duct cancer, bladder cancer, bronchus cancer, nervous tissue cancer, gall bladder cancer, stomach cancer, salivary gland cancer, esophagus cancer, small intestine cancer, cervix cancer, colon cancer, rectum cancer, liver cancer, ovary cancer, pancreas cancer, melanoma, pituitary adenoma, secretory adenoma, synovial sarcoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia.

[0027] In an embodiment, the infectious disease is selected from the group consisting of viral infection, bacterial infection, fungal infection and parasitic infection.

[0028] In another aspect, the present application provides the use of bleomycin or an analogue thereof for the preparation of an immune enhancer that enhances MHC-I and / or MHC-II dependent immune responses.

[0029] In an embodiment, the bleomycin is selected from the group consisting of BLM A2, BLM B2, BLM A5 and combinations thereof. In a preferred embodiment, the bleomycin is a mixture of BLM A2 and BLM B2.

[0030] In an embodiment, the bleomycin analogue is selected from the group consisting of peliomycin, rachelmycin, liblomycin, zorbamycin, corchmycin and combinations thereof.

[0031] In an embodiment, the immune enhancer enhances the anti-tumor effect of an anti-tumor immunotherapeutic agent involved in MHC-I and / or MHC-II dependent anti-tumor immune responses.

[0032] In an embodiment, the immune enhancer enhances an anti-infectious immune response, the infection being selected from the group consisting of viral infection, bacterial infection, fungal infection and parasitic infection. BRIEF DESCRIPTION OF DRAWINGS

[0033] Embodiments of the present application are described in detail by way of reference only to the attached drawings wherein:

[0034] Figure 1 shows the expression level of basal HLA-A in K562, DB, SK-BR-3, SU-DHL-4, T47D and BT549 cells by Western blotting. GAPDH expression level was used as an internal control.

[0035] Figure 2A shows the expression level of HLA-A / B / C on the cell membrane surface of SU-DHL-4 cells treated with different concentrations of bleomycin for 48 h, detected by flow cytometry. The example histogram (upper panel) and the statistical chart (lower panel) are shown. **p<0.01, ***p<0.001. Con, control.

[0036] Figure 2B shows the expression level of HLA-A / B / C on the cell membrane surface of SU-DHL-4 cells treated with 10 μΜ bleomycin for different time, detected by flow cytometry. The example histogram (upper panel) and the statistical chart (lower panel) are shown. ***p<0.001. Con, control.

[0037] Figure 2C shows the expression level of HLA-A detected by Western blotting of SU-DHL-4 cells treated with different concentrations of bleomycin for 48 h (upper panel), or treated with 10 μΜ bleomycin for different time (lower panel). GAPDH expression level was used as internal control.

[0038] Figure 2D shows the mRNA expression level of HLA-A, HLA-B, HLA-C, B2M, TAP1, TAP2, TAPBP, PSMB8 and PSMB9 detected by RT-PCR of SU-DHL-4 cells treated with 10 μΜ bleomycin for 48 h. *p<0.05, **p<0.01, ***p<0.001.

[0039] Figure 3A shows the expression level of HLA-A detected by Western blotting of K562 cells treated with different concentrations of bleomycin for 48 h. GAPDH expression level was used as internal control.

[0040] Figure 3B shows the expression level of HLA-A / B / C on the cell membrane surface of K562 cells treated with different concentrations of bleomycin for 48 h, detected by flow cytometry. The example histogram (upper panel) and the statistical chart (lower panel) are shown. ***p<0.001. Con, control.

[0041] Figure 4 shows the expression level of HLA-DR / DP / DQ on the cell membrane surface of SU-DHL-4 cells treated with different concentrations of bleomycin for 48 h, detected by flow cytometry. The example histogram (upper panel) and the statistical chart (lower panel) are shown. ***p<0.001. Con, control.

[0042] Figure 5 shows the results of B16F10 or B16OVA cells treated with different concentrations of bleomycin and co-cultured with OT-I T cells. Shown in the figure are crystal violet staining (upper panel) and apoptosis analyzed by flow cytometry (lower panel).

[0043] Figure 6 shows the secretion of IFNy detected by ELISA after B16F10 or B16OVA cells were co-cultured with OT-I T cells. ***p<0.001.

[0044] Figure 7 shows the effect of bleomycin in the mouse adoptive cell therapy model. Figure 7A shows the experimental flow chart. Figure 7B shows the change of body weight of mice in different groups during the experiment. Figure 7C shows the tumor weight of mice in different groups at the end of the experiment. Figure 7D shows the change of tumor volume of mice in different groups during the experiment. *p<0.05, ***p<0.001 compared with the control group. ###p<0.001 compared with the corresponding group.

[0045] Figure 8 shows the expression level of B2m in tumor tissues of mice in different groups detected by Western blot. Figure 8A shows the Western blot figure. Figure 8B shows the results of quantification of protein bands in Figure 8A. ***p<0.001.

[0046] Figure 9 shows the mRNA expression levels of Gzmb, Ifng and Prf1 in tumor tissues of mice detected by RT-PCR. *p<0.05.

[0047] Figure 10 shows the expression level of Granzyme B in tumor tissues of mice in different groups detected by immunofluorescence. DAPI staining shows the cell nucleus.

[0048] Figure 11 shows the survival curve of mice in different treatment groups.

[0049] Figure 12 shows the effect of bleomycin in promoting the infiltration of OT-I T cells into tumor tissues in the mouse adoptive cell therapy model. Shown is the content of OT-I T cells in the single cell suspension of tumor tissues of mice treated with bleomycin (combination group) or not treated with bleomycin (OT-I T cell group) three days after receiving tail vein infusion of OT-I T cells (D14) analyzed by flow cytometry.

[0050] Figure 13 shows the effect of bleomycin on the killing activity of CD8 +Results of T cell co-culture. Fig. 13A shows the results of crystal violet staining. Fig. 13B shows the secretion of IFNy detected by ELISA. Fig. 13C shows the cell viability detected by CellTiter Glo Luminescent Cell Viability Assay Kit. **p<0.01, ***p<0.001.

[0051] Fig. 14 shows the change of tumor volume of mice in different treatment groups during the experiment in B16F10 mouse xenograft tumor model. ***p<0.001 compared with the control group. ###p<0.001 compared with the corresponding group.

[0052] Fig. 15 shows the tumor weight of mice in different treatment groups at the end of the experiment in B16F10 mouse xenograft tumor model. **p<0.01, ***p<0.001 compared with the control group. #p<0.05, ###p<0.001 compared with the corresponding group.

[0053] Fig. 16 shows the change of body weight of mice in different treatment groups during the experiment in B16F10 mouse xenograft tumor model.

[0054] Fig. 17 shows the results of analyzing the expression level of HLA-A by Western blot after primary bladder cancer cell lines were treated with different concentrations of bleomycin for 48h. Con, control.

[0055] Fig. 18 shows the cell viability detected by CellTiter Glo Luminescent Cell Viability Assay Kit after primary bladder cancer cell lines were treated with different concentrations of bleomycin for 72h.

[0056] Fig. 19 shows the results of analyzing TILs cells isolated and cultured from two bladder cancer patient tumor samples, BCC101 and BCC102, by multicolor flow cytometry.

[0057] Fig. 20 shows the results of detecting cell viability by CellTiter Glo Luminescent Cell Viability Assay Kit after primary bladder cancer cells derived from BCC101 and BCC102 were co-cultured with corresponding autologous TILs cells for about 24h. *p<0.05, **p<0.01.

[0058] Fig. 21 shows the results of analyzing cell apoptosis by crystal violet staining or flow cytometry after primary bladder cancer cells derived from BCC101 were co-cultured with autologous TILs cells for about 24h. Fig. 21A shows the results of analyzing cell apoptosis by crystal violet staining (upper panel) or flow cytometry (lower panel). Fig. 21B shows the rate of cell apoptosis analyzed by flow cytometry. ***p<0.001. DETAILED DESCRIPTION

[0059] General Definitions and Terminology

[0060] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless indicated otherwise. Also, the terms and techniques employed herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunological procedures and laboratory procedures are those well known and commonly used in the corresponding art. In addition, the definitions and explanations of the relevant terms are provided below for better understanding of the present application.

[0061] As used herein, the terms "comprising," "including," "containing," and "having" are inclusive and do not exclude additional, unrecited elements, steps, or components. The term "consisting of" excludes any element, step, or component not specified. The term "consisting essentially of" means a range limited to the specified elements, steps, or components, plus an optional presence of elements, steps, or components that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. It is understood that the terms "consisting essentially of" and "consisting of" are encompassed within the meaning of the term "comprising."

[0062] As used herein, the term "optional" or "optionally" means that the subsequent description is applicable or can be applicable. This term encompasses instances where the event occurs and instances where the event does not occur.

[0063] As used herein, the conjunctive term "and / or" between elements of a plurality of recited elements is to be understood to include the individual and combined options.

[0064] Unless otherwise indicated, any numerical values or value ranges, such as concentrations or concentration ranges, are to be understood to be modified by the term "about" in any context. Thus, a numerical value typically includes ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerals within the range, including integers and fractions, unless the context clearly indicates otherwise.

[0065] As used herein, an "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope of an antigen through at least one antigen binding site. In this context, the definition of antibody encompasses antigen-binding fragments. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (e.g., produced by chemical or biological conjugation), enzymatically processed, or recombinantly produced. Antibodies include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass (e.g., IgG2a and IgG2b). Antibodies can be "monovalent," "bivalent," "trivalent," or "tetravalent" or more, referring to the number of antigen binding sites they contain. Antibodies can be monoclonal or polyclonal, and are preferably monoclonal.

[0066] As used herein, an "antigen-binding fragment" refers to a portion of a full-length antibody that is less than full-length but at least contains a portion of the variable region of the full-length antibody (e.g., contains one or more CDRs and / or one or more antigen binding sites) and thus retains at least part of the full-length antibody's ability to specifically bind an antigen. Examples of antigen-binding fragments include, but are not limited to, sdAbs (e.g., variable domains of heavy chain antibodies), Fv, scFv, dsFv, scdsFv, scDb (single chain diabody), Fab, scFab, Fab', F(ab')2, diabodies, Fd, and Fd' fragments, and other fragments (e.g., containing modifications).

[0067] As used herein, the term "antibody-drug conjugate" refers to a drug comprising a cytotoxic drug conjugated to an antibody that targets the cytotoxic drug to target cells expressing an antigen through specific binding of the antibody to the antigen.

[0068] As used herein, to "enhance" or "induce" an immune response means to increase the magnitude and / or efficiency of an immune response, or to prolong the duration of an immune response.

[0069] An "immune response" or "immune response" refers to any response of the immune system of a vertebrate to an immunogenic substance (e.g., a polypeptide or polynucleotide or fragment). Exemplary immune responses include local and systemic cellular immunity and humoral immunity, such as cytotoxic T lymphocyte (CTL) (including CD8 + antigen-specific induction of CTLs), helper T cells (including T cell proliferation and cytokine release), and B cell-mediated immune responses.

[0070] As used herein, the term "pharmaceutical combination" refers to the combination of two or more active agents in one form. It will be appreciated that these agents can be in admixture or in separate forms, such as in a composition or mixture, or in separate compartments of a kit or in different kits. For example, the agents in a pharmaceutical combination can be formulated as one pharmaceutical composition for simultaneous administration. Alternatively, each agent can be formulated separately as independent pharmaceutical compositions, which can be administered simultaneously, sequentially, or separately. The agents in a pharmaceutical combination can be administered in a simultaneous, sequential, overlapping, alternating, parallel, or any other therapeutic schedule when the various agents are administered as part of a therapeutic regimen. The active ingredients are exemplified by bleomycin or an analog thereof or one or more additional cancer therapeutic agents (e.g., an anti-tumor immunotherapeutic agent described herein).

[0071] As used herein, the term "pharmaceutical composition" refers to an active ingredient, optionally in combination with one or more pharmaceutically acceptable components (e.g., but not limited to, a carrier). In addition to the active ingredient, a pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers. It will be appreciated by those skilled in the art that bleomycin or an analog thereof and one or more additional cancer therapeutic agents (e.g., an anti-tumor immunotherapeutic agent described herein) can be formulated in one pharmaceutical composition, which can be used, for example, for simultaneous administration. Alternatively, bleomycin or an analog thereof and one or more additional cancer therapeutic agents (e.g., an anti-tumor immunotherapeutic agent described herein) can be formulated in various pharmaceutical compositions, which can be used, for example, for simultaneous, sequential, or separate administration. It will also be appreciated by those skilled in the art that a pharmaceutical composition can independently and optionally comprise one or more pharmaceutically acceptable carriers.

[0072] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, sodium chloride, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerin), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA (Sucrose-Phosphate-Glutamate-Albumin), sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0073] As used herein, the term "effective amount" refers to that amount of a drug or pharmaceutical agent or a combination which is sufficient to effect the desired effect. The effective amount can be determined alone, and depends on the age and general condition of the recipient and the particular active ingredient. The effective amount in a particular case can be determined by a person skilled in the art by routine testing. When two or more pharmaceutical agents are used in combination, for example, in the form of a claimed pharmaceutical combination, the effective amount also refers to the effective amount of each agent to exert a synergistic effect. For example, for the treatment of a tumor, an "effective amount" of bleomycin or a pharmaceutical combination comprising the same preferably inhibits tumor cell growth or tumor growth in a subject by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received treatment. The efficacy of inhibiting tumor growth can be assessed using routine tumor animal models, for example, spontaneous tumor, induced tumor and transplanted tumor animal models. Alternatively, the ability to inhibit cell growth can also be examined using in vitro test methods well known in the art. An effective amount of bleomycin or a pharmaceutical combination comprising the same is capable of reducing tumor size, or otherwise ameliorating the symptoms in a subject (e.g., preventing and / or treating metastasis or recurrence). The effective amount can be given in one or more administrations.

[0074] The term "active ingredient" or "therapeutic agent" refers to a chemical entity that is useful in the treatment or prevention of a target disorder, disease or condition. Unless otherwise indicated, the agents in the pharmaceutical combination (e.g., anti-tumor immunotherapeutic agents) are commercially available or can be readily synthesized or obtained according to routine means in the art.

[0075] As used herein, the definition of "subject" includes human and non-human subjects, such as experimental animals (e.g., mice, rabbits, rats, and non-human primates), preferably humans.

[0076] As used herein, "therapeutic effect" refers to an effect on altering the symptoms of a disease or disorder as a result of treatment of a subject, typically an effect of improving or eliminating the symptoms of the disease or disorder.

[0077] As used herein, "therapeutically effective amount" refers to that amount of an agent, compound or composition comprising one or more active agents which is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.

[0078] Bleomycin and analogs thereof

[0079] In one aspect, the present application provides the use of bleomycin or an analog thereof in the manufacture of an immune enhancer which enhances MHC-I and / or MHC-II dependent immune responses.

[0080] Without wishing to be bound by any theory, the present inventors have surprisingly found that bleomycin or an analogue thereof is capable of increasing the expression level of MHC-I and MHC-II molecules on the surface of a cell, such as a tumor cell. According to one specific aspect of the present application, bleomycin or an analogue thereof enhances CD4 + T cell and / or CD8 + T cell and / or CD8 + T cell and / or CD8+T cell mediated immune response.

[0081] In some embodiments, the immune enhancing agent enhances an anti-tumor immune response. Accordingly, in one specific aspect, the present application provides the use of bleomycin or an analogue thereof in the manufacture of an anti-tumor immune enhancing agent. In some embodiments, bleomycin enhances a T cell and tumor infiltrating lymphocyte mediated anti-tumor immune response by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a tumor cell. In some embodiments, bleomycin enhances a T cell engaging bispecific antibody mediated anti-tumor immune response by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a tumor cell. In some embodiments, bleomycin enhances the anti-tumor effect of an immune checkpoint inhibitor, such as an anti-PD-Ll antibody, by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a tumor cell. + T cell and tumor infiltrating lymphocyte) mediated anti-tumor immune response. In some embodiments, bleomycin enhances a T cell engaging bispecific antibody mediated anti-tumor immune response by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a tumor cell. In some embodiments, bleomycin enhances the anti-tumor effect of an immune checkpoint inhibitor, such as an anti-PD-Ll antibody, by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a tumor cell.

[0082] In some embodiments, the immune enhancing agent enhances an anti-infective immune response. Accordingly, in another specific aspect, the present application also provides the use of bleomycin or an analogue thereof in the manufacture of an anti-infective immune enhancing agent. In some embodiments, bleomycin enhances a MHC-I and / or MHC-II dependent anti-infective immune response by increasing the expression level of MHC-I and / or MHC-II molecules on the surface of a pathogen infected cell. In one embodiment, the infection is selected from the group consisting of viral infection, bacterial infection, fungal infection and parasitic infection.

[0083] As used herein, bleomycin (BLM) is a glycopeptide produced by Streptomyces verticillus that belongs to the family of bleomycin antibiotics. BLM causes sequence-specific double-stranded DNA cleavage through a metal-dependent mechanism. BLM comprises four functional domains: (1) a metal-binding domain comprising pyrimidoblamic acid and beta-hydroxyhistidine; (2) a DNA-binding domain consisting of a C-terminal amine tail that interacts with a bithiazole moiety and a pyrimidine amine ring; (3) a linker region connecting the metal-binding domain and the DNA-binding domain; and (4) a disaccharide moiety responsible for cell selectivity and DNA cleavage activity (Chen, J. and Stubbe, J., Nat Rev Cancer 5, 102-112 (2005); and Coughlin JM et al., Biochemistry. 2014 Nov 11; 53(44):6901-9). As used herein, the definition of “bleomycin” encompasses addition salts of bleomycin with acids, such as its hydrochloride and sulfate salts. Examples of bleomycin include, but are not limited to, BLM A2 (CAS: 11056-06-7), BLM B2 (CAS: 9060-10-0), BLM A5 (CAS: 11116-32-8), and mixtures thereof.

[0084] In one embodiment, the bleomycin is selected from the group consisting of BLM A2, BLM B2, BLM A5 (pingyangmycin), and combinations thereof. In a preferred embodiment, the bleomycin is a mixture of BLM A2 and BLM B2 (e.g., bleomycin sulfate, Blenoxane). In another preferred embodiment, the bleomycin is BLM A5 (e.g., pingyangmycin hydrochloride).

[0085] The bleomycin antibiotic family also includes bleomycin analogs such as Peplomycin (CAS: 68247-85-8), Tallysomycin (CAS: 65057-90-1), Liblomycin (CAS: 88266-67-5), Zorbamycin (ZBM; CAS: 11056-20-5), and Phleomycin (CAS: 11006-33-0), among others. Like bleomycin, bleomycin analogs are also glycopeptide antibiotics that induce DNA damage through a metal-dependent mechanism and have antitumor activity. As used herein, the definition of “bleomycin analog” includes addition salts of bleomycin analogs with acids, such as its hydrochloride and sulfate salts. In one embodiment, the bleomycin analog is selected from the group consisting of Peplomycin (e.g., Peplomycin sulfate), Tallysomycin, Liblomycin, Zorbamycin, Phleomycin, and combinations thereof.

[0086] Pharmaceutical combinations and kits

[0087] In another aspect, the present application provides a pharmaceutical combination comprising a bleomycin, a bleomycin analog, or a combination thereof, and an immunotherapeutic agent (in particular an antitumor immunotherapeutic agent). The pharmaceutical combination of the present application can be in the form of a pharmaceutical composition or a kit.

[0088] As used herein, the term “immunotherapeutic agent” refers to a therapeutic agent that treats a disease (e.g., cancer and infection) by inducing or enhancing an immune response. Diseases that can be treated by an immunotherapeutic agent include, but are not limited to, cancer, viral infection, bacterial infection, fungal infection, and parasitic infection.

[0089] As used herein, the term “antitumor immunotherapeutic agent” refers to a therapeutic agent that treats cancer by inducing or enhancing an immune response. In some embodiments, an immunotherapeutic agent (e.g., an antitumor immunotherapeutic agent) can increase the expression and / or activity of an immune activator. In some embodiments, an immunotherapeutic agent (e.g., an antitumor immunotherapeutic agent) can decrease the expression and / or activity of an immune suppressor. In some embodiments, an immunotherapeutic agent (e.g., an antitumor immunotherapeutic agent) can recruit and / or enhance the activity of an immune cell.

[0090] According to some aspects of the application, bleomycin can facilitate the anti-tumor or anti-infective effect of an immunotherapeutic agent by enhancing the expression of MHC-I and / or MHC-II molecules on the surface of a target cell (e.g., a tumor cell or a pathogen-infected cell). In an embodiment, the immunotherapeutic agent (e.g., an anti-tumor immunotherapeutic agent) is involved in an MHC-I and / or MHC-II dependent immune response (e.g., an anti-tumor immune response). The immunotherapeutic agent (e.g., an anti-tumor therapeutic agent) can be directly (e.g., mediate) or indirectly (e.g., induce or enhance) involved in the immune response (e.g., an anti-tumor immune response). In some embodiments, the immunotherapeutic agent (e.g., an anti-tumor therapeutic agent) mediates, induces or enhances an MHC-I and / or MHC-II dependent immune response (e.g., an anti-tumor immune response).

[0091] An MHC-I dependent immune response (e.g., an anti-tumor immune response) can for example include, but is not limited to: immune reactions mediated by CD8 + cytotoxic T lymphocytes (CD8 + CTLs, also commonly known as cytotoxic CD8 + T cells) (e.g., by expressing antigen-specific T cell receptors (TCRs)) (including for example CTL activation, clonal expansion and enhancement of CTL effector functions), antibody (e.g., antibodies targeting peptide-MHC-I complexes (pMHC-I)) mediated effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP)) and cytotoxic effects (e.g., by immunotoxins targeting pMHC-I).

[0092] An MHC-II dependent immune response (e.g., an anti-tumor immune response) can for example include, but is not limited to: immune reactions mediated by CD4 + cytotoxic T lymphocytes (CD4 + CTLs, also commonly known as cytotoxic CD4 + T cells) (e.g., by expressing antigen-specific T cell receptors (TCRs)) (including for example CTL activation, clonal expansion and enhancement of CTL effector functions).

[0093] In a particular aspect, the immunotherapeutic agent (e.g., an anti-tumor immunotherapeutic agent), such as TCR-like antibodies and immunotoxins comprising the same, CTLs, TCR engineered T cells (TCR-Ts), chimeric antigen receptor T cells (CAR-Ts) and tumor infiltrating lymphocytes (TILs) and the like, can mediate an immune response (e.g., an anti-tumor immune response). In another particular aspect, the immunotherapeutic agent (e.g., an anti-tumor immunotherapeutic agent), such as immune checkpoint inhibitors and tumor neoantigen-based vaccines and the like, can induce or enhance an immune response (e.g., an anti-tumor immune response), for example by activating CD8+ T cells kill target cells (e.g., tumor cells), for example, by perforin (Prfl) and granzyme B (Gzmb) to induce target cell apoptosis. In addition, activated CD8 + T cells can also produce various cytokines, including IFNy and IL-2. IFNy can further increase MHC class-I molecule expression, increasing CD8 + T cell recognition of target cells.

[0094] As used herein, the term “MHC-I” or “MHC-II” refers to any native MHC-I (major histocompatibility complex-I) or MHC-II (major histocompatibility complex-II) from any vertebrate (including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Human MHC-I is also known as human leukocyte antigen I (HLA-I). Human MHC-II is also known as human leukocyte antigen II (HLA-II).

[0095] The expression level of MHC-I or HLA-I can be assessed by determining the expression level of any HLA-I gene or pseudogene (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L), or haplotype thereof. The expression level of MHC-I molecules can be assessed by detecting the expression level of, for example, HLA heavy chains (e.g., HLA-A, HLA-B, and HLA-C) and B2M. In addition, TAP1, TAP2, TAPBP, PSMB8, and PSMB9 are all closely related to MHC-I expression levels and antigen presentation mediated thereby. In some embodiments, one or more of bleomycin and analogs thereof can increase or at least increase by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% the surface expression of MHC-I molecules (e.g., HLA-A, HLA-B, HLA-C, and / or B2M) and optionally the expression of TAP1, TAP2, TAPBP, PSMB8, and / or PSMB9 in about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of tumor cells relative to control cells or a control population of cells.

[0096] HLA-Class II genes include the HLA-D family, which includes, among others, HLA-DR, HLA-DP, and HLA-DQ. The level of MHC-II or HLA-II expression can be assessed by detecting the expression level of HLA-DR, HLA-DP, and HLA-DQ. In some embodiments, one or more of bleomycin and analogs thereof can increase the surface expression of MHC-II molecules (e.g., HLA-DR, HLA-DP, and HLA-DQ) in about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of tumor cells relative to control cells or a control population of cells, or by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%.

[0097] The immunotherapeutic agents (e.g., anti-tumor immunotherapeutic agents) that can be used in the present application can be, for example, antibodies, antibody-drug conjugates, and immune cells that participate in an immune response (e.g., an anti-tumor immune response) in an MHC-I dependent or MHC-II dependent manner.

[0098] In some embodiments, the immunotherapeutic agent targets an antigenic peptide-MHC-I complex (pMHC-I) and / or an antigenic peptide-MHC-II complex (pMHC-II), the antigenic peptide selected from the group consisting of a tumor antigenic peptide, a viral antigenic peptide, a bacterial antigenic peptide, a fungal antigenic peptide, and a parasitic antigenic peptide.

[0099] In some embodiments, the antigenic peptide is a tumor antigenic peptide. In some embodiments, the tumor antigenic peptide comprises an MHC-I restricted epitope selected from the group consisting of KRAS, MAGE1, gp100, hTERT, NY-ESO-1, hCGbeta, Her2 / Neu, Melan-A / MART-1, TARP, p53, p68, MIF, Proteinase 3 (PR1), WT1, HA-1, PRAME, CEA, MAGE-A3, and MAGE-A4. In some embodiments, the tumor antigenic peptide comprises an MHC-I restricted epitope selected from the group consisting of KRAS, gp100, and p53.

[0100] In some embodiments, the immunotherapeutic agent is an anti-tumor immunotherapeutic agent. In some embodiments, the anti-tumor immunotherapeutic agent targets (e.g., through specific binding between an antibody (or TCR)-antigen) an antigenic peptide-MHC-I complex (pMHC-I) on the surface of a tumor cell, which comprises an MHC-I restricted epitope in a tumor-specific antigen. As used herein, “MHC-I restricted epitope” refers to a peptide sequence recognized by CTLs in conjunction with MHC-I. MHC-I restricted epitopes in tumor-specific antigens include, but are not limited to, those listed in Table 1 and Table 2. In an embodiment, the MHC-I restricted epitope in a tumor-specific antigen is selected from MHC-I restricted antigenic epitopes in the following proteins: KRAS, MAGE1, gp100, hTERT, NY-ESO-1, hCGβ, Her2 / Neu, Melan-A / MART-1, TARP, p53, p68, MIF, Proteinase 3 (PR1), WT1, HA-1, PRAME, CEA, MAGE-A3, and MAGE-A4.

[0101] In some embodiments, the anti-tumor immunotherapeutic agent targets an antigenic peptide-MHC-II complex (pMHC-II) on the surface of a tumor cell, which comprises an MHC-II restricted epitope in a tumor-specific antigen. As used herein, “MHC-II restricted epitope” refers to a peptide sequence recognized by CTLs in conjunction with MHC-II.

[0102] In some embodiments, the anti-tumor immunotherapeutic agent is selected from an antibody, an antibody-drug conjugate, an immune cell, and combinations thereof. In some embodiments, the anti-tumor immunotherapeutic agent can be or comprise an immune cell, e.g., is or comprises a T cell receptor engineered immune cell. In some embodiments, the anti-tumor immunotherapeutic agent is selected from a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), a T cell receptor engineered T cell (TCR-T), and combinations thereof. Anti-tumor immunotherapeutic agents that can be used in the pharmaceutical combination of the present application can include those based on tumor-specific antigens, in particular tumor neoantigens (see, e.g., Xie, N., Shen, G., Gao, W. et al. Sig Transduct Target Ther 8, 9 (2023)). The term “tumor neoantigen” refers to a neoantigen that is present in a tumor cell or tissue but not in the corresponding normal cell or tissue. Methods for identifying neoantigens are well known in the art, see, e.g., CN108601731A. Descriptions of tumor neoantigens can be found, e.g., in CN115968299A (multiple myeloma), CN113573729A (prostate cancer), and CN115515623A (ovarian cancer), which are incorporated herein in their entirety.

[0103] To generate a desired anti-tumor immune response, the anti-tumor immunotherapeutic can particularly advantageously comprise a tumor antigen-specific TCR or TCR-like antibody. “Tumor antigen-specific” as used with respect to a TCR, a TCR-like antibody, or an anti-tumor immunotherapeutic comprising the same (e.g., a tumor antigen-specific antibody-drug conjugate and an immune cell) refers to the TCR or TCR-like antibody specifically recognizing (“targeting”) an antigenic peptide-MHC-I complex or an antigenic peptide-MHC-II complex on the surface of a tumor cell, the antigenic peptide-MHC-I complex comprising an MHC-I restricted epitope in a tumor-specific antigen, in particular a tumor neoantigen, the antigenic peptide-MHC-II complex comprising an MHC-II restricted epitope in a tumor-specific antigen, in particular a tumor neoantigen. In an embodiment, the anti-tumor immunotherapeutic comprises a T cell receptor (TCR) or a TCR-like antibody.

[0104] In some embodiments, the immunotherapeutic (e.g., an anti-tumor immunotherapeutic) comprises a T cell receptor (TCR). As used herein, the term “T cell receptor” or “TCR” refers to a membrane protein complex that participates in T cell activation in response to antigen presentation. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. The TCR consists of a heterodimer of alpha and beta chains, although in some cells the TCR consists of gamma and delta chains. Each chain consists of two extracellular domains, a variable domain (antigen binding) and a constant domain. In this context, the term “T cell receptor” or “TCR” encompasses full-length native TCR polypeptides and TCR artificial constructs. Examples of immunotherapeutics (e.g., anti-tumor immunotherapeutics) comprising a TCR can include native or artificially engineered immune cells, such as cytotoxic T lymphocytes (CTLs) and T cell receptor engineered T cells (TCR-Ts). In a particular embodiment, the anti-tumor immunotherapeutic is selected from the group consisting of cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), T cell receptor engineered T cells (TCR-Ts), and combinations thereof. In some embodiments, the TCR targets a peptide-MHC-I complex on the surface of a tumor cell, the peptide-MHC-I complex comprising an MHC-I restricted epitope in a tumor-specific antigen, in particular a tumor neoantigen. MHC-I restricted epitopes that can be targeted by a TCR (or an immune cell comprising the same) include, but are not limited to, those listed in Table 2.

[0105] In an embodiment, the anti-tumor immunotherapeutic comprises a TCR-like antibody. As used herein, the term “TCR-like antibody” or “TCR-mimicking antibody” generally refers to an antibody capable of recognizing a peptide-MHC complex (e.g., a peptide-MHC-I complex, pMHC-I) on the surface of an infected cell or on the surface of a tumor cell. As used herein, the meaning of “TCR-like antibody” includes antigen-binding fragments. TCR-like antibodies can be screened or generated by known methods, e.g., by phage library or hybridoma. Non-limiting examples of anti-tumor immunotherapeutics comprising a TCR-like antibody can include: TCR-like antibodies and fusion proteins, antibody-drug conjugates (e.g., immunotoxins), and immune cells comprising a TCR-like antibody. In some embodiments, the anti-tumor immunotherapeutic comprising a TCR-like antibody is selected from the group consisting of a TCR-like antibody, a T cell engager, an immunotoxin, a chimeric antigen receptor T cell, and combinations thereof. In an embodiment, the anti-tumor immunotherapeutic is a TCR-like antibody. In an embodiment, the anti-tumor immunotherapeutic is a T cell engager comprising a TCR-like antibody. In an embodiment, the anti-tumor immunotherapeutic is an immunotoxin comprising a TCR-like antibody. In an embodiment, the anti-tumor immunotherapeutic is a T cell expressing a chimeric antigen receptor comprising a TCR-like antibody. In some embodiments, the TCR-like antibody targets a peptide-MHC-I complex on the surface of a tumor cell, the peptide-MHC-I complex comprising an MHC-I restricted epitope in a tumor-specific antigen (in particular, a tumor neoantigen). pMHC-I (e.g., tumor antigen peptide / HLA-A complex) that a TCR-like antibody (or an anti-tumor immunotherapeutic comprising the same) can target include, but are not limited to: p53 R175H / HLA-A*02:01 complex (e.g., antibody H2, which can be combined with an anti-CD3 antibody to obtain a T cell engaging bispecific antibody useful in the present application, e.g., H2-scDb in the form of a single-chain diabody), KRAS G12V / HLA-A*03:01 (e.g., antibody V2, which can be combined with an anti-CD3 antibody to obtain a T cell engaging bispecific antibody useful in the present application, e.g., V2-scDb in the form of a single-chain diabody), gp100 peptide-HLA-A*02:01 (e.g., tebentafusp, a T cell engager that specifically recognizes gp100 peptide-HLA-A*02:01 and CD3, for the treatment of, e.g., uveal melanoma). Examples of other tumor surface antigen peptide / HLA-A complexes can also be found in Table 1.

[0106] TCR-like antibodies that can be used include, but are not limited to, those described in He Q et al, J Hematol Oncol. 2019 Sep 14; 12(1): 99, the entirety of which is incorporated herein by reference. Examples of particularly preferred TCR-like antibodies or pMHC-I that they target can also be found in Table 1.

[0107] Table 1

[0108] TCR-like antibodies (e.g., antigen-binding fragments of TCR-like antibodies, such as scFv, scFab, Fab, and scDb) can be prepared into T cell engagers (e.g., BiTEs), CARs, antibody-drug conjugates (e.g., by fusing TCR-like antibodies with cytotoxic agents (e.g., immunotoxins) or antibody formats with ADCC / ADCP / CDC effector functions (e.g., by fusion to IgGl Fc or IgG2 Fc) to obtain anti-tumor immunotherapeutics as described herein. Thus, in accordance with certain aspects of the present application, non-limiting examples of particularly useful anti-tumor immunotherapeutics include: TCR-like antibodies, T cell engagers comprising TCR-like antibodies, immunotoxins comprising TCR-like antibodies, and chimeric antigen receptor T cells (CAR-Ts).

[0109] As used herein, “immunotoxin” refers to an immunotherapeutic agent that contains a targeting moiety (e.g., an antigen-binding fragment, such as a Fab) fused to a cytotoxic agent, particularly a toxic protein. Toxic proteins that can be used to prepare immunotoxins can include, for example, diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin A (PE), ricin, and granzyme. Immunotoxins that can be used in the pharmaceutical combinations of the present application can be obtained, for example, by fusing a TCR-like antibody as described herein with a cytotoxic agent. Immunotoxins that can be used in the pharmaceutical combinations of the present application include, but are not limited to, immunotoxins that target the following pMHC-I: gp100 / HLA-A*0201 (for treating, e.g., melanoma, see, e.g., Denkberg G, et al., J Immunol. 2003; 171(5):2197-207, and Klechevsky E, et al., Cancer Res. 2008; 68(15):6360-7), TARP / HLA-A*0201 (for treating, e.g., breast and prostate cancer, see, e.g., Klechevsky E, et al., Cancer Res. 2008; 68(15):6360-7), and Melan-A / MART-1 / HLA-A*0201 (for treating, e.g., melanoma, see, e.g., Epel M, et al., Eur J Immunol. 2008; 38(6): 1706-20).

[0110] As used herein, “T cell engager (TCE)” refers to a polypeptide construct, often a bispecific antibody (also known as “bispecific T cell engager (BiTE)” or “T cell engaging bispecific antibody”), that can simultaneously bind a tumor associated antigen (TAA) or neoantigen / HLA-A complex on a tumor cell and a CD3 epitope on a T cell, which results in the T cell being activated and producing a cytotoxic effect on the tumor cell. In some embodiments, the T cell engager comprises a TCR-like antibody. Examples of T cell engagers comprising a TCR-like antibody include, but are not limited to, antibody H2-scDb (see Hsiue EH et al., Targeting a neoantigen derived from a common TP53 mutation. Science. 2021 Mar 5; 371(6533): eabc8697), antibody V2-scDb (see Douglass J et al., Bispecific antibodies targeting mutant RAS neoantigens. Sci Immunol. 2021 Mar 1; 6(57): eabd5515), and tebentafusp.

[0111] In an embodiment, the T cell engager is a T cell engaging bispecific antibody. In a preferred embodiment, the T cell engaging bispecific antibody comprises a TCR mimicking antibody (also known as “TCR-like antibody”) that targets CD3 and a tumor cell surface neoantigen / HLA-A complex. In a preferred embodiment, the T cell engaging bispecific antibody is selected from antibody H2-scDb, antibody V2-scDb, and tebentafusp. In an embodiment, the T cell engaging bispecific antibody is tebentafusp. In an embodiment, the T cell engaging bispecific antibody is antibody H2-scDb. In an embodiment, the T cell engaging bispecific antibody is antibody V2-scDb.

[0112] In an embodiment, antibody H2-scDb comprises a scFv that specifically binds p53 R175Ha first portion of an / HLA-A*02:01 complex and a second portion that specifically binds CD3, wherein the first portion comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, and the second portion comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0113] The immunotherapeutic agent (e.g., anti-tumor immunotherapeutic agent) in the pharmaceutical combination of the present application can also be an immune cell involved in immune response through MHC-I and / or MHC-II mediated antigen presentation. The immune cell can be or comprise a CD8 + T cell or a CD4 + T cell. Such immune cells can include tumor antigen specific CTLs and TILs (which comprise tumor antigen specific CTLs) isolated from a cancer patient. Tumor antigen specific TCRs or TCR-like antibodies can also be introduced into suitable immune cells to obtain engineered immune cells, such as TCR-T and CAR-T. The immune cells useful in the pharmaceutical combination of the present application can be commonly used in Adoptive Cell Transfer Therapy (ACT). Adoptive Cell Transfer Therapy refers to the reinfusion of immune cells (e.g., T cells) into a patient after in vitro culture and / or engineering, so as to eliminate tumor cells. The immune cells for Adoptive Immune Cell Therapy can be autologous or allogeneic. In a preferred embodiment, the anti-tumor immunotherapeutic agent is selected from the group consisting of cytotoxic T lymphocytes (CTLs), T cell receptor engineered T cells (TCR-Ts), chimeric antigen receptor (CAR) T cells, and tumor infiltrating lymphocytes (TILs).

[0114] As used herein, the term “T cell receptor engineered T cell” or “TCR-T” refers to any T cell having a T cell receptor that is heterologous to the T cell. In the present application, particularly useful TCR-Ts include engineered T cells expressing TCRs that specifically recognize tumor antigen MART-1 (for treatment of metastatic melanoma, see, e.g., Rohaan MW et al., Immunooncol Technol. 2022 Jun 18; 15: 100089), Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) (for treatment of, e.g., EBV-associated cancers, see, e.g., CN113166224A), and NY-ESO-1 (for solid tumor treatment, see, e.g., Gnjatic S et al., Adv Cancer Res. 2006; 95: 1-30). TCR-Ts can also target pMHC-I as shown in Table 2. Examples of TCR-Ts that can be used in the present application also include, but are not limited to, those described in Ping Y et al., Protein Cell. 2018 Mar; 9(3): 254-266, which is incorporated herein in its entirety.

[0115] Table 2

[0116] The term“chimeric antigen receptor T cell” or“CAR-T cell” refers to a lymphocyte expressing a chimeric antigen receptor. The term“chimeric antigen receptor” or“CAR” has its art- conventional meaning and refers to an artificially constructed protein or polypeptide containing an antigen binding domain of an antibody (e.g. scFv) linked to a T cell signaling domain. The features of a CAR can include the ability to redirect T cell specificity and reactivity to a selected target, taking advantage of the antigen binding properties of monoclonal antibodies. Generally, a CAR can comprise an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. It has been reported in the prior art that TCR-like antibodies can be converted into CAR structures to mediate T cell specificity for tumor cell lysis. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. Strategies to design and produce such CARs are well known in the art, see for example Bonini and Mondino, Eur. J. Immunol. 2015 (19), Srivastava and Riddell, Trends Immunol. 2015 (20), Jensen and Riddell, Curr. Opin. Immunol. 2015 (21), and Gill and June, Immunol. Rev. 2015 (22). CAR-T cells that can be used in the pharmaceutical combination of the present application can include for example T cells expressing a CAR that specifically recognizes the following antigen / HLA-A complex: PR1 / HLA-A2 (for the treatment of e.g. myeloid leukemia, see for example Ma Q et al., Cytotherapy. 2016 Aug;18(8):985-994), MAGE1 / HLA-A*0101 (for the treatment of e.g. melanoma, see for example Willemsen RA, et al. Gene Ther. 2001;8(21): 1601-8, and Chames P, et al. J Immunol. 2002;169(2): 1110-8) and gp100 / HLA-A*0201 (for the treatment of e.g. melanoma, see for example Zhang G, et al. Sci Rep. 2014;4:3571.).

[0117] “tumor infiltrating lymphocytes” or“TILs” are found in the tumor tissue stroma and are composed of CD8 + T cells, CD4 + T cells, B cells, NK cells, macrophages and neutrophils, among others, where direct killing of tumor cells is mainly through CD8 +T cell effectors. TILs therapy involves isolation of TILs from a patient's own tumor tissue, in vitro stimulation and expansion, screening, and reinfusion of immune cells with anti-tumor effect to the patient. Methods for obtaining TILs are known in the art, for example by preparing a single cell suspension from a subject's tumor tissue. Further purification of the desired TILs type from the cell suspension can also be performed using methods known in the art, for example by selecting for appropriate cell surface markers (by FACS or magnetic beads). Descriptions of such methods can be found, for example, in The Handbook of Experimental Immunology, Vol 1-4, (D.N. Weir, editor) and Flow Cytometry and Cell Sorting (A. Radbruch, editor, Springer Verlag, 2000).

[0118] Without being bound by any theory, it is believed that immune checkpoint signaling is inhibitory to immune cells (e.g. CD8 +T cell)-mediated anti-tumor immune responses, an important mechanism that leads to immune escape of tumor cells. The prior art has demonstrated the potential of immune checkpoint inhibitors in treating various types of tumors. According to certain aspects of the present application, bleomycin or its analogs can facilitate the anti-tumor effect of immune checkpoint inhibitors by enhancing the expression of MHC-I and / or MHC-II molecules on the surface of tumor cells. It is believed that immune checkpoint inhibitors also play an important role in anti-infective immune responses. Thus, the immunotherapeutic agent (e.g., anti-tumor immunotherapeutic agent) in the pharmaceutical combination of the present application can also be an immune checkpoint inhibitor. Non-limiting examples of immune checkpoint inhibitors can include antibodies that block the immune inhibitory activities of PD-L1, PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT. Methods to obtain these antibodies are well known in the art. In an embodiment, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, and an anti-TIGIT antibody. Methods to obtain these antibodies are well known in the art. In an embodiment, the immune checkpoint inhibitor is an anti-PD-L1 antibody. Non-limiting examples of anti-PD-L1 antibodies include Atezolizumab, Durvalumab, Avelumab, BMS-936559. Further descriptions of anti-PD-L1 antibodies can be found, for example, in US US7943743B2, US9580507B2, WO2011066389, and WO2012145493. In yet another embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody. Non-limiting examples of anti-PD-1 antibodies include Pembrolizumab, Nivolumab, Cemiplimab, Retifanlimab, Toripalimab, and Dostarlimab. In another embodiment, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. Non-limiting examples of anti-CTLA-4 antibodies include Ipilimumab, Tremelimuniab, AGEN-1884, and ATOR-1015. In another embodiment, the immune checkpoint inhibitor is an anti-TIM-3 antibody. Non-limiting examples of anti-TIM-3 antibodies include TSR-022 and LY3321367. In another embodiment, the immune checkpoint inhibitor is an anti-LAG-3 antibody. Non-limiting examples of anti-LAG-3 antibodies include Relatlimab, Fianlimab, and Ieramilimab. Non-limiting examples of anti-TIGIT antibodies include BMS-986207, Tiragolumab, Ociperlimab, Vibostolimab, Domvanalimab, COM902 (CGEN-15137), and Etigilimab.

[0119] The immunotherapeutic agent in the pharmaceutical combination of the present application can also be a vaccine. As used herein, the term “vaccine” refers to a substance capable of being processed into an antigenic peptide, e.g., MHC-I or MHC-II presented antigenic peptide, in a subject to induce an immune response in the subject. For example, the anti-tumor immunotherapeutic agent can be a neoantigen-based cancer vaccine. Tumor neoantigens are present in cancer patients, but are confined to cancer cells only. It is believed that administration of a neoantigen-based vaccine to a cancer patient can activate the immune system to activate CD8 + and CD4 + T cells to thereby destroy cancer cells. An individualized neoantigen vaccine can train the immune system to recognize and kill cancer cells that present neoantigens. However, the success of this process can depend on several factors, including antigen presentation by MHC molecules. According to certain aspects of the present application, bleomycin or its analogs can facilitate MHC-I and / or MHC-II restricted presentation of tumor neoantigens by enhancing the expression of MHC-I and / or MHC-II molecules on the surface of tumor cells. The terms “neoantigen-based cancer vaccine” or “neoantigen vaccine” can be used interchangeably and are vaccine constructs based on one or more tumor neoantigens, e.g., a plurality of tumor neoantigens. Examples of neoantigen vaccines include, but are not limited to, those described in Biswas N et al., Front Immunol. 2023 Feb 9; 14: 1105420, the entirety of which is incorporated herein by reference.

[0120] The present application further provides a pharmaceutical composition comprising the pharmaceutical combination of the present application and a pharmaceutically acceptable carrier. In a particular embodiment, the pharmaceutical composition of the present application comprises bleomycin, a bleomycin analog or a combination thereof, an anti-tumor immunotherapeutic agent, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition of the present application comprises a first pharmaceutical composition comprising bleomycin, a bleomycin analog or a combination thereof, and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising an anti-tumor immunotherapeutic agent and a pharmaceutically acceptable carrier.

[0121] Pharmaceutically acceptable carriers can include, but are not limited to, diluents, binding and gluing agents, lubricants, disintegrants, preservatives, vehicles, dispersing agents, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like), solubilizers, gelling agents, emollients, solvents (e.g., water, alcohol, acetic acid, and sugar syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., non-ionic surfactants such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents, and emulsifying agents. For compositions comprising an antibody or antibody conjugate, suitable carriers can be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), non-ionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer), or combinations thereof.

[0122] The pharmaceutical compositions provided herein can be in a variety of dosage forms, including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or topical (e.g., by injection or infusion) administration. For compositions comprising an antibody or antibody conjugate, preferred dosage forms can generally be, for example, injectable solutions and lyophilized powders.

[0123] Bleomycin or the pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local). It will be understood by those skilled in the art that the exact dosage administered will depend upon a variety of factors, such as the metabolic pharmacokinetic properties of the pharmaceutical composition, the duration of treatment, the rate of excretion of the particular compound, the purpose of the treatment, the route of administration, and the condition of the subject, such as the age, health, weight, sex, diet, medical history of the patient, and other factors well known in the medical arts. Methods of administration can be, for example, injection or infusion.

[0124] In yet another aspect, the present application provides a kit comprising a pharmaceutical combination or a pharmaceutical composition of the present application. The kit can further comprise a suitable container, such as an ampoule. The pharmaceutical combination of the present application can be provided in different compartments of the kit or in different kits. In some embodiments, the kit further comprises a device for administration. The kit can further comprise a label indicating intended use of the contents of the kit and / or methods of use. The term "label" includes any written or recorded material that is provided with or is associated with a kit, or is otherwise provided with the kit.

[0125] The present application also provides use of a pharmaceutical combination, a kit or a pharmaceutical composition of the present application in the manufacture of a medicament for the treatment and / or prevention of a cancer or an infectious disease.

[0126] The terms "cancer", "tumor" and "cancerous tumor" have the same meaning herein and include, but are not limited to, solid tumors and blood cancers. Exemplary solid tumors include, but are not limited to, head and neck squamous cell carcinoma, breast cancer, bone cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer), adrenal cancer (e.g., adrenocortical tumor), bile duct cancer, bladder cancer, bronchus cancer, nervous tissue cancer (including neuronal and neuroglial tumors), gall bladder cancer, stomach cancer, salivary gland cancer, esophagus cancer, small intestine cancer, uterine cervical cancer, colon cancer, rectum cancer, liver cancer, ovarian cancer, pancreatic cancer, melanoma, pituitary adenoma, and a secretory adenoma. Exemplary blood cancers include, but are not limited to, lymphomas and leukemias. Exemplary lymphomas include, but are not limited to, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., diffuse large cell lymphoma, HTLV-1 associated T-cell lymphoma, lymphoma nodal peripheral T-cell lymphoma, extranodal peripheral T-cell lymphoma, central nervous system lymphoma, and AIDS-related lymphoma). Exemplary leukemias include, but are not limited to, acute and chronic types of lymphocytic and myelogenous leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia).

[0127] In view of the present application, one skilled in the art is readily able to determine the cancer that can be treated and / or prevented using the pharmaceutical combination, kit, or pharmaceutical composition of the present application. In one embodiment, the cancer is a cancer in which the cancer cells have low expression of MHC-I and / or MHC-II. In some embodiments, the cancer is a cancer in which the cancer cells increase expression of MHC-I and / or MHC-II in response to treatment with bleomycin or an analog thereof. In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer is a hematological cancer. In one embodiment, the cancer is a solid tumor in which the cancer cells have low expression of MHC-I and / or MHC-II. In one embodiment, the cancer is a hematological cancer in which the cancer cells have low expression of MHC-I and / or MHC-II. Many cancer cells (e.g., head and neck squamous cell carcinoma cells, breast cancer cells, colon cancer cells, ovarian cancer cells, melanoma cells, Hodgkin lymphoma cells, non-small cell lung cancer cells, and bladder cancer cells) show downregulated MHC-I cell surface expression. In some embodiments, low expression of MHC-I includes a decrease in the expression level of a gene selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, HLA-L, and B2M. In some embodiments, the cancer is a cancer in which the cancer cells have low expression of a MHC-I associated gene selected from the group consisting of TAP1, TAP2, TAPBP, PSMB8, and PSMB9. In some embodiments, low expression of MHC-II includes a decrease in the expression level of a gene selected from the group consisting of HLA-DR, HLA-DP, and HLA-DQ.

[0128] In one embodiment, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, bone cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer), adrenal cancer (e.g., adrenocortical tumor), bile duct cancer, bladder cancer, bronchial cancer, nervous tissue cancer (including neuronal and glial tumors), gall bladder cancer, gastric cancer, salivary gland cancer, esophageal cancer, small intestine cancer, cervical cancer, colon cancer, rectal cancer, liver cancer, ovarian cancer, pancreatic cancer, melanoma, pituitary adenoma, secretory adenoma, synovial sarcoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia. In a particularly preferred embodiment, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, synovial sarcoma, breast cancer, colon cancer, rectal cancer, ovarian cancer, melanoma, Hodgkin lymphoma, non-small cell lung cancer, and bladder cancer.

[0129] “Infectious disease” refers to a disease caused by infection with a pathogen, including but not limited to viral infection, bacterial infection, fungal infection, and parasitic infection. In some embodiments, the infectious disease is selected from the group consisting of viral infection, bacterial infection, fungal infection, and parasitic infection.

[0130] Methods of enhancing an immune response

[0131] In one aspect, the present application provides a method for enhancing an immune response in a subject comprising administering to the subject an effective amount of bleomycin, a bleomycin analog, or a combination thereof.

[0132] In a specific aspect, the present application provides a method for enhancing a T cell mediated immune response in a subject comprising administering to the subject an effective amount of bleomycin, a bleomycin analog, or a combination thereof. In a preferred embodiment, the T cell is a CD8 + T cell.

[0133] In another aspect, the present application provides a method for treating or preventing cancer comprising administering to a subject in need thereof an effective amount of a pharmaceutical combination or a pharmaceutical composition of the present application.

[0134] In a specific aspect, the present application provides a method for treating or preventing cancer comprising administering to a subject in need thereof an effective amount of bleomycin, a bleomycin analog, or a combination thereof, and an anti-tumor immunotherapeutic agent as described herein.

[0135] In one embodiment, the method comprises administering an effective amount of bleomycin, a bleomycin analog, or a combination thereof, prior to administering an effective amount of the anti-tumor immunotherapeutic agent. In one embodiment, the method comprises administering an effective amount of bleomycin, a bleomycin analog, or a combination thereof, and the anti-tumor immunotherapeutic agent simultaneously. In one embodiment, the method comprises administering an effective amount of the anti-tumor immunotherapeutic agent prior to administering an effective amount of bleomycin, a bleomycin analog, or a combination thereof.

[0136] In some embodiments, the effective amount of bleomycin, a bleomycin analog, or a combination thereof does not result in lung toxicity, e.g., pulmonary fibrosis. The effective amount of bleomycin, a bleomycin analog, or a combination thereof can be administered in a single or multiple dosages. For example, the effective amount of bleomycin, a bleomycin analog, or a combination thereof can be administered in one, two, three or more dosages per day. In one embodiment, the effective amount of bleomycin comprises 15 mg bleomycin / kg body weight or less per day, e.g., 15, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5 mg bleomycin / kg body weight per day. In a preferred embodiment, the effective amount of bleomycin comprises 1-5 mg bleomycin / kg body weight per day. In one embodiment, the effective amount of bleomycin comprises 2-4 mg bleomycin / kg body weight per day. In a particular embodiment, the effective amount of bleomycin comprises 3 mg bleomycin / kg body weight per day. Beneficial effects

[0137] According to some aspects of the present application, bleomycin or an analog thereof is capable of increasing the expression level of MHC-I and / or MHC-II on the surface of a cell. In a particular aspect, bleomycin promotes an anti-tumor immune response by increasing the expression level of MHC-I and / or MHC-II on the surface of a cancer cell. For example, bleomycin or an analog thereof is capable of antigen-dependent activation of CD8+ T cells by increasing the expression level of MHC-I on the surface of a cancer cell, thereby promoting a CD8+ T cell-mediated immune response. For example, BLM enhances the antigen-specific immune response of CD8+ T cells (OT-I T cells) to tumor cells, increases the sensitivity of bladder cancer patient-derived tumor cells to autologous tumor-infiltrating lymphocytes (TILs)-mediated cytotoxicity. Low-dose bleomycin in combination with immunotherapeutic agents (e.g., TCR-engineered T cells, T cell engaging bispecific antibodies H2-scDb, tumor-infiltrating lymphocytes, and anti-PD-L1 antibodies) inhibits tumor growth by promoting the killing of tumor cells by cytotoxic T cells and increasing the infiltration of tumor lymphocytes, and avoids possible side effects such as lung toxicity, demonstrating excellent clinical treatment potential. In summary, the pharmaceutical combination of the present application can exhibit an anti-tumor synergistic effect and good safety.

[0138] Examples

[0139] The technical solutions of the present application will be further described below through specific examples. It should be noted that these examples are only exemplary and not a limitation on the scope of protection of the present application. Unless otherwise specified, the materials and instruments used in the following examples are commercially available or prepared according to methods known in the art. The experimental methods in the following examples without specific conditions are carried out according to the conventional methods and conditions, or according to the instructions of the goods.

[0140] Materials and methods of example 1

[0141] 1.1 Materials and equipment

[0142] The following antibodies were used for protein immunoblotting: B2m (abcam: ab75853; 1:50000); GAPDH (Proteintech: 60004, Cat No. 60004-1-Ig; 1:50000); HLA-A (ABclonal: A11406; 1:10000); goat anti-rabbit secondary antibody (Jackson Immunology 111-035-003).

[0143] The following antibodies were used for flow cytometry: APC-Cy7 Mouse Anti-Human CD45 (2D1) (BD Pharmingen: 561863); FITC Mouse Anti-Human CD3 (HIT3a) (BD Pharmingen: 561802); BV605 Mouse Anti-Human CD4 (RPA-T4) (BD Pharmingen: 562658); Alexa Fluor 700 Mouse Anti-Human CD8 (RPA-T8) (BD Pharmingen: 561453); BV510 Mouse Anti-Human CD56 (NCAM16.2) (BD Pharmingen: 563041); PerCP-Cy5.5 Rat Anti-CD11b (M1 / 70) (BD Pharmingen: 561114); PE-Cy7 Mouse Anti-Human CD16 (3G8) (BD Pharmingen: 557744); APC Mouse Anti-human PD-1 (Biolegend: 621610); APC anti-human HLA-A / B / C (Biolegend: 311410); APC anti-human HLA-DR / DP / DQ (Biolegend: 361713).

[0144] Bleomycin (Taoka Bio: T6116) stock solution was dissolved in PBS with a storage concentration of 10 mmol / L.

[0145] Ovalbumin (OVA) peptide (MCE) stock solution was dissolved in ddH2O with a storage concentration of 1 mg / mL.

[0146] 1.2 Methods

[0147] Animals

[0148] Before the experiment, female C57BL / 6J mice (6-8 weeks) (Shanghai Jiesijie Experimental Animal Co., Ltd.) were placed in the animal room for three days to adapt to the growth. During the tumor experiment, the body weight and tumor volume of the mice were monitored and recorded every other day (tumor volume V = (a x b 2 ) / 2, where a and b are the long and short diameters of the tumor, respectively).

[0149] Cells

[0150] All cells were cultured in a 37 °C, 5% CO2 constant temperature sterile incubator (Thermo Scientific Forma Series II Water Jacket).

[0151] SU-DHL-4 cells, B16F10 cells (purchased from the Chinese Academy of Sciences Cell Bank) and B16OVA cells (constructed based on B16F10) were cultured in the following medium: 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U / mL penicillin and 100 U / mL streptomycin (Gibco).

[0152] SK-BR-3 cells (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in the following medium: DMEM medium (Gibco) supplemented with 20% FBS, 100 U / mL penicillin and 100 U / mL streptomycin.

[0153] OT-I T cells and CD8+ T cells were cultured in the following medium: 1640 medium supplemented with 10% medium, 100 U / mL penicillin and 100 U / mL streptomycin, 2 mM L-glutamine (Gibco), 10 mM HEPES (Gibco), non-essential amino acids (1x) (Gibco) and 50 mM β-mercaptoethanol (Sigma).

[0154] Tumor infiltrating lymphocytes (TILs) were cultured in the following medium: 1640 medium supplemented with inactivated 10% AB serum (GemCell TMHuman Serum AB: Gemini Bio), 100 U / mL penicillin and 100 U / mL streptomycin, 2 mM L-glutamine, 10 mM HEPES (Gibco), non-essential amino acids (1x) (Gibco), 50 mM β-mercaptoethanol, and 6000 IU / mL recombinant human IL-2 protein (Peprotech).

[0155] Purification of bispecific antibody

[0156] The plasmid expressing bispecific antibody H2-scDb is p8400_IL-2_H2-ScDb_6xHIS tag plasmid, which contains the signal peptide sequence of IL-2, followed by the bispecific antibody H2-scDb sequence connected by the enzyme cutting site, and the 6xHIS tag at the C-terminus of the antibody sequence for subsequent purification. The bispecific antibody coding sequence is synthesized by Kingsriver Biotech Co., Ltd. In general, 1.2 mg of plasmid is transfected into 1L 293-F cells by PEI, and after three days of transfection, the cell supernatant is collected. First, the collected cell supernatant is centrifuged at 12000 rpm at 4°C for 60 min. Continue to collect the cell supernatant after centrifugation, and filter through 0.45 μM filter paper. Then purified by Ni-NTA agarose.

[0157] Crystal violet staining

[0158] Fix the cells with methanol at room temperature for 20 min. Wash with ddH2O for 3 times. Add crystal violet staining solution and stain at room temperature for 20 min. Wash with ddH2O for 3 times, 2 min each time. Observe the staining results under a microscope (Olympus IX711) and take pictures.

[0159] Western blot

[0160] The specific method is as follows: the protein sample extracted with RIPA lysis buffer is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (electrophoresis instrument: BioRad PowerPac HC), and then transferred to an NC membrane (Pall Corporation, item number: 66485) or a PVDF membrane (Millipore Immunobilon) (eBlot L1 protein transfer instrument (Gensheng Biotechnology Co., Ltd.)); after the transfer is completed, the membrane is placed in an appropriate amount of blocking solution (1xPBST containing 5% BSA or skimmed milk powder), and blocked at room temperature for 1 h; the first antibody is diluted with the blocking solution, and incubated at room temperature for 2-4 h or at 4°C overnight; after the incubation is completed, the membrane is washed with 1xPBST (1xPBS+0.1% Tween-20) for 3 times, each time for 5 minutes; then the second antibody with HRP labeling is diluted with 1xPBST for 1 h at room temperature, and the membrane is washed with 1xPBST for 3 times, each time for 5 minutes; the required color developing substrate (1:1) is prepared, and developed and imaged in a chemiluminescence instrument (integrated chemiluminescence imaging instrument and chemiluminescence liquid (Shanghai Qinxing Biotechnology Co., Ltd.)).

[0161] RNA extraction, reverse transcription and real-time fluorescent quantitative PCR (qPCR)

[0162] According to the manufacturer's instructions, total RNA of cells is extracted using GeneJET RNA Purification Kit (Thermo Fisher Scientific (China) Co., Ltd.), and the extracted RNA is reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Nanjing Novelpia Biotech Co., Ltd., item number: R223). Then according to the manufacturer's instructions, using ChamQ Universal SYBR qPCR Master Mix (Nanjing Novelpia Biotech Co., Ltd., item number: Q711), using the obtained cDNA as a template, using gene-specific primers (synthesized by Shanghai Sangon) for real-time fluorescent quantitative PCR (real-time fluorescent PCR instrument (Roche)).

[0163] The PCR primer sequences used in Example 2 are shown in the following table:

[0164] The PCR primer sequences used in Example 4 are shown in the following table:

[0165] According to the ΔΔCT method, the relative amount of mRNA is calculated, GAPDH or Actb is used as an internal reference, the mRNA level of the control group (samples without bleomycin treatment) is set to 1, and the relative amount is obtained by comparing each treatment group with the control group.

[0166] Enzyme-linked immunosorbent assay (ELISA)

[0167] The secretion of IFN-γ was detected using Mouse IFN-γ ELISA Set (BD Phar mingen) according to the manufacturer's instruction. The specific method is as follows: the capture antibody was diluted with coating buffer (Na2CO3 / NaHCO3 solution, containing 0.3565 g sodium bicarbonate and 0.0795 g sodium carbonate per 50 mL, pH 7.4) at a ratio of 1:250, 100 μL of the diluted solution was added to each well of a 96-well plate for coating; the liquid in the plate was discarded, and the plate was washed 5 times with 1 × PBS + 0.05% Tween-20 (washing solution); 200 μL of Assay Dilute (PBS + 10% FBS) was added, and incubated at room temperature for 1 hour; the liquid in the plate was discarded, and the plate was washed 5 times with 1 × PBST; 100 μL of sample was added to each well, and incubated at room temperature for 2 hours; the liquid in the plate was discarded, and the plate was washed 5 times with 1 × PBST; 100 μL of Working Detector (detection antibody 1:250 and SAV-HRP 1:250 added to Assay Dilute) was added, and incubated at room temperature for 1 hour; the liquid in the plate was discarded, and the plate was washed 10 times with 1 × PBST; 100 μL of substrate solution (tetramethylbenzidine (TMB) and hydrogen peroxide) (EL-TMB color developing kit, purchased from Shengong Bioengineering (Shanghai) Co., Ltd., item number: C520026) was added: 5 mL of solution A + 250 μL of solution B + 1 μL of solution C, and incubated at room temperature for 20 min; 50 μL of solution D (EL-TMB color developing kit) was added to terminate the reaction, and the absorbance (OD450) at 450 nm was read using an enzyme reader (EnSpire Multilabel Reader (PerkinElmer)).

[0168] Cell viability detection

[0169] CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) was used to detect cell viability according to the manufacturer's instructions. The specific method is as follows: the CellTiter-Glo buffer and CellTiter-Glo substrate lyophilized powder were taken out of the refrigerator and equilibrated to room temperature, then the CellTiter-Glo buffer was added to the CellTiter-Glo substrate lyophilized powder according to the instructions, and the mixture was shaken to dissolve completely and then stored for later use; after the cell co-culture treatment was completed, the cells were equilibrated at room temperature for 30 min; the pre-prepared CellTiter-Glo reagent was added to the test wells at a ratio of CellTiter-Glo reagent: culture medium supernatant = 1:1; shake for two minutes to fully lyse the cells; after incubation at room temperature for ten minutes, the luminescence signal value in the test well was detected by a microplate reader.

[0170] Flow cytometry

[0171] Apoptosis: Annexin V, 633 Apoptosis Detection Kit (Dojindo) was used to detect apoptosis by flow cytometry according to the manufacturer's instructions. The specific method is as follows: the culture medium supernatant was aspirated into a 15 mL centrifuge tube, and the tumor cells in the trypsinized 12-well plate were stopped by the culture medium supernatant previously placed in the 15 mL tube. The residual adherent cells in the plate were blown off, and all the cells were transferred to the 15 mL centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and 1 mL of flow washing solution (PBS + 2% FBS) was added to resuspend the cells and transferred to a 1.5 mL ep tube. Then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded; the cells were resuspended with 50 mL of flow washing solution (PBS + 2% FBS), and a certain amount of anti-mouse CD45 fluorescent primary antibody was added according to the antibody instructions, and the same amount of isotype control antibody was added to the control tube; incubate at 4°C for 30 min in the dark, resuspend with 1 mL of flow washing solution (PBS + 2% FBS), centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; add 100 μL Annexin binding buffer, and add anti-Annexin V-FITC and propidium iodide (PI) according to the antibody instructions; incubate at room temperature for 10 min in the dark, add 200 μL Annexin binding buffer, and use a flow cytometer to detect within one hour.

[0172] Cell surface expression of HLA-A / B / C: Bleomycin-treated SU-DHL-4 tumor cells were collected in 1.5 mL ep tubes; the cells were centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; 1 mL of flow washing solution (PBS + 2% FBS) was added to resuspend the cells, which were centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; 50 μL of flow washing solution was used to resuspend the cells, and a certain amount of anti-human HLA-A / B / C fluorescent primary antibody (Biolegend) was added according to the requirements of the antibody instructions; the control tube was added with the corresponding isotype control antibody; 4°C, avoid light, incubate for 30 min, add 1 mL of flow washing solution to resuspend the cells, centrifuge the cells at 1000 rpm for 5 min, and aspirate the supernatant; after washing, 200 μL of flow washing solution was added to resuspend the cells, and the flow cytometer was used to detect the HLA-A / B / C on the surface of the tumor cells.

[0173] Mouse tumor tissue immune cell infiltration analysis: The cut mouse tumor tissue was shaken in a 37°C constant temperature incubator shaker for 30-40 min after being shaken in 1.5 mL of 1640 solution containing 1 mg / mL collagenase and 10 μg / mL DNA enzyme, and then 5 mL of PBS containing 2% FBS was added for neutralization; a 200-mesh screen was used for filtration, and the filtered cell suspension was centrifuged; the supernatant was discarded, 1 mL of red blood cell lysis solution was added, and the lysis was performed at room temperature for 3 min, then 9 mL of PBS containing 2% FBS was added for neutralization; then 10 mL of PBS containing 2% FBS was added for resuspension, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; 100 μL of PBS containing 2% FBS was used to resuspend the cells, and a certain amount of anti-CD45.1 fluorescent primary antibody was added according to the requirements of the antibody instructions, and the control tube was added with the corresponding isotype control antibody; 4°C, avoid light, incubate for 30 min, add 1 mL of PBS containing 2% FBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, and aspirate the supernatant; after washing, 250 μL of PBS containing 2% FBS was added to resuspend the cells, and the flow cytometer was used for detection.

[0174] Immunofluorescence staining

[0175] The paraffin section is sequentially soaked in xylene, anhydrous ethanol, 85% ethanol, 75% ethanol and ddH2O to remove paraffin; the citrate antigen repair buffer is heated to boiling with a microwave oven, and the deparaffinized section is gently placed in the citrate antigen repair buffer, during which the temperature is intermittently heated to maintain between 95-98°C, and incubated for 10 min and then naturally cooled; the sample is incubated with 5% blank goat serum at 37°C for 30 min for blocking; the blocking solution is removed, and the diluted primary antibody is added and incubated at 4°C overnight; rewarming: the sample is placed at room temperature and rewarming for 15 min; the antibody working solution is removed, washed once with buffer TBST for 5 min; washed three times with buffer TBS, each for 5 min; the diluted secondary antibody is added and incubated at room temperature for 1 h in the dark; the secondary antibody working solution is removed, washed once with buffer TBST for 5 min; washed three times with buffer TBS, each for 5 min; DAPI working solution is added on the sample, in the dark, at room temperature, and incubated for 10 min; the DAPI working solution is removed, washed once with buffer TBST for 5 min; washed three times with buffer TBS, each for 5 min; after adding the anti-fluorescence fading mounting medium, the sample is observed under a fluorescence microscope and the image is collected.

[0176] Isolation of human PBMCs

[0177] Fresh peripheral blood is aseptically extracted into an ACD / EDTA / heparin anticoagulation tube; an equal volume of PBS is added to the anticoagulation tube containing whole blood to dilute the blood, and the test tube is turned several times to mix the blood and buffer; Ficoll Paque Plus (GE Healthcare) is added to the bottom of a 50 mL centrifuge tube, and the amount of Ficoll-Paque added is adjusted according to the volume of the diluted blood sample, ensuring that Ficoll-Paque:diluted blood = 3:4; the diluted blood sample is carefully layered on the Ficoll-Paque solution; centrifugation is performed at 2400 rpm for 20 min at room temperature, ensuring that the speed is reduced to 0; the uppermost plasma is aspirated, and the mononuclear cell layer is not disturbed at the interface; the white membrane layer is transferred to a new centrifuge tube, about 3 times the volume of PBS is added to the centrifuge tube, and the cell precipitate is uniformly blown with a pipette to fully wash the cells, and centrifugation is performed at 2400 rpm for 10 min at room temperature; the supernatant is discarded, and the precipitate is resuspended with PBS and centrifuged at 1200 rpm for 5 min at room temperature; if red blood cells are mixed, add 1 mL of red cell lysing solution, repeatedly blow with a gun head, and count the time for 3-5 min; add a large amount of PBS to terminate the red cell lysis, and centrifuge at 1000 rpm for 5 min at room temperature; discard the supernatant, resuspend the precipitate with culture medium, and the PBMC cells obtained at this time are obtained.

[0178] Irradiation of human PBMCs

[0179] The collected PBMCs were resuspended with medium and placed in 50 mL centrifuge tubes; the centrifuge tubes were transported to the Shanghai Radiation Institute for irradiation at a dose of 50 Gy; after irradiation, the PBMCs were counted; the irradiated PBMCs were generally not recommended to be frozen and needed to be immediately subjected to the next step of the experiment.

[0180] Isolation of human CD8+T cells

[0181] The EasySep Human CD8+T Cell Enrichment Kit (STEMCELL) was used to isolate CD8+T cells from the prepared PBMC cells according to the manufacturer's instructions. TM The isolated CD8+T cells were cultured and expanded with CD8+T cell medium.

[0182] Isolation and culture of tumor infiltrating lymphocytes (TILs)

[0183] The method for isolating and culturing TILs from surgical samples, puncture samples or cystoscope samples (solid samples of tumor tissues) is as follows: after the tumor sample was transported back to the laboratory from the ice box, the tissue was cut into small pieces with sterilized scissors and forceps, and the small pieces were placed in a 24-well plate to which TIL culture medium had been added; the expanded TILs were closely observed to ensure that the medium was changed at least once a week; after 3-4 weeks of initial expansion, each well was numbered as F1, F2, F3,..., F12, and a small amount of cells in each well were taken for multicolor flow detection; the primary tumor cells were co-cultured with the TILs in each well, and only the TILs with killing effect were left; at this time, the TILs could be frozen or directly subjected to the next step of expansion culture; the TILs rapid expansion culture medium contained 1x10 3 -1x10 4 TILs and 100-300 times of irradiated PBMC cells, 30 ng / mL of anti-CD3 antibody and 3000 IU / mL of recombinant human IL2; on the 5th day, the 9th day and the 12th day, additional recombinant human IL2 was supplemented; on the 7th day, the expanded TILs were changed; about two weeks later, the expanded TILs were collected for subsequent co-culture experiments.

[0184] Co-culture of TILs with primary bladder cancer cells

[0185] Specific method as follows: 1E5 primary bladder cancer tumor cells were plated in cell culture plate; after the cells adhered, the primary bladder cancer tumor cells were pretreated with a certain concentration of bleomycin for 48h; after the expanded tumor infiltrating lymphocytes of different numbers were collected, centrifuged to remove supernatant, resuspended with culture medium, and then counted; after the culture medium containing drugs was removed, washed once with PBS, and a certain proportion of tumor infiltrating lymphocytes (TILs: tumor cells = 3:1) were added; after co-culture for about 24-48h, tumor cell apoptosis was detected by flow cytometry or crystal violet staining.

[0186] Extraction and pre-activation of OT-I T cells

[0187] OT-I T cells were extracted from the livers of 6-8 week old OT-I female mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J, 003831; see Hogquist KA et al., Cell. 1994 Jan 14; 76(1): 17-27). The specific method is as follows: the mouse spleen was ground into a single cell suspension; centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; 1 mL of ice-cold red blood cell lysis solution was added, inverted to mix, and lysed for 3 min; 9 mL of PBS was added and inverted to mix, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; 10 mL of PBS was added again to resuspend, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated; the cells were resuspended with OT-I T cell growth medium, and the cell density was adjusted to 5x10 6 / mL, and an OVA peptide solution with a final concentration of 100 ng / mL was added; after 48 hours of culture, 2-3 times OT-I T cell growth medium was added, and recombinant mouse IL-2 protein (STEMCELL) with a final concentration of 10 ng / mL was added to the medium; after 48h of culture, the OT-I T cells at this time could be used for co-culture experiments (or after further ficoll density gradient centrifugation to remove dead cells, for reinfusion experiments).

[0188] Example 2 Bleomycin increases the expression level of MHC-I and MHC-II on the surface of tumor cells

[0189] To verify the ability of bleomycin to increase the expression level of MHC-I on the surface of tumor cells, the expression level of background HLA-A in K562 (a human chronic myeloid leukemia cell), DB (a diffuse large B-cell lymphoma cell), SK-BR-3 (a human breast cancer cell), SU-DHL-4 (a human lymphoma cell), T47D (a human breast ductal carcinoma cell) and BT549 cell (a human breast ductal carcinoma cell) was first detected, and the results are shown in Figure 1. As shown in Figure 1, the expression level of background HLA-A in SK-BR-3, DB and K562 cells was essentially undetectable, the expression level of background HLA-A in SU-DHL-4 cells was relatively low, and the expression level of background HLA-A in T47D and BT549 cells was relatively high.

[0190] The expression level of MHC-I molecules in SU-DHL-4 cells treated with bleomycin was detected by flow cytometry, Western blotting and qPCR, and the results are shown in Figures 2A-2D.

[0191] As shown in Figures 2A and 2B, after the SU-DHL-4 tumor cells were treated with bleomycin, the expression level of MHC-I on the surface of the tumor cells was increased, and there was a concentration-dependent and time-dependent trend, as detected by flow cytometry. As shown in Figure 2C, bleomycin increased the protein level of HLA-A in SU-DHL-4 tumor cells, and there was a concentration-dependent and time-dependent trend. As shown in Figure 2D, the transcription level of MHC-I related genes (including: HLA-A; HLA-B; HLA-C; B2M; TAP1; TAP2; TAPBP; PSMB8; PSMB9) was increased.

[0192] Therefore, it was further detected whether bleomycin could increase the expression level of MHC-I in K562 cells, and the results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, even in K562 cells with a very low expression level of HLA-A, bleomycin could dose-dependently increase the expression level of MHC-I.

[0193] It was further studied whether bleomycin could increase the expression level of MHC-II on the surface of tumor cells. After SU-DHL-4 cells were treated with different concentrations of bleomycin, the expression level of HLA-DR / DP / DQ in the tumor cells was detected by flow cytometry. The results are shown in Figure 4. As shown in Figure 4, after the SU-DHL-4 tumor cells were treated with bleomycin, the expression level of MHC-II on the surface of the tumor cells was increased, and there was a dose-dependent trend.

[0194] The above results show that bleomycin can increase the expression level of MHC-I and MHC-II on the surface of tumor cells.

[0195] Example 3: Bleomycin promotes the killing of tumor cells by antigen-specific T cells.

[0196] The clinical potential of bleomycin combined with antigen-specific T cells for tumor treatment was investigated in a co-culture experiment of mouse melanoma cells B16OVA and OT-I T cells. B16OVA cells were B16F10 cells expressing ovalbumin (OVA). OT-I transgenic mice expressed OVA-sensitive antigens on CD8+ T cells. 257-264 (SIINFEKL) peptide-specific T cell receptor (TCR). Therefore, OT-I T cells isolated from OT-I transgenic mice can antigen-specifically target and kill B16OVA cells.

[0197] B16F10 or B16OVA cells were treated with bleomycin and then co-cultured with OT-I T cells. Tumor cell apoptosis was detected by crystal violet staining and flow cytometry, and IFNγ secreted by OT-I T cells was detected by ELISA. The specific method is as follows: B16F10 or B16OVA cells in good growth condition were cultured at 3.5 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 12-well plates; then treated with 0 μM (PBS), 1.25 μM, or 2.5 μM bleomycin for 48 h; subsequently, the culture supernatant was discarded, cells were gently rinsed with PBS, and OT-IT cells were added at a ratio of 1:1 to 5:1. After co-culturing for approximately 24 h, the killing effect on tumor cells was observed under a microscope and photographed. A portion of the cells from the 12-well plates were treated as follows: the culture supernatant was transferred to a clean 15 mL centrifuge tube, and a small amount was transferred from the supernatant to a 1.5 mL ep tube for ELISA detection of IFNγ secretion; then, the tumor cells in the 12-well plates were digested with trypsin (Gibco), and digestion was terminated with the culture supernatant previously placed in the 15 mL tube. The remaining adherent cells in the plate were then pipetted, and all cells were transferred to 15 mL centrifuge tubes. Apoptosis was then detected by flow cytometry. Another portion of the cells in the 12-well plate were treated as follows: the culture supernatant was discarded, and the cells were gently washed with PBS until no OT-1 T cells or apoptotic tumor cells remained in the culture plate. The remaining tumor cells were detected by crystal violet staining. The results are shown in Figures 5 and 6.

[0198] As shown in Figure 5, B16F10 cells did not undergo apoptosis when co-cultured with OT-I T cells. Even after bleomycin pretreatment, B16F10 cells did not exhibit significant apoptosis when co-cultured with OT-I T cells. However, when B16OVA cells were co-cultured with OT-I T cells, B16OVA cells underwent more significant apoptosis, with an apoptosis rate of approximately 20%. After bleomycin pretreatment, B16OVA cells further underwent significant apoptosis when co-cultured with OT-I T cells, with apoptosis rates of approximately 35% and 55% in the 1.25 μM and 2.5 μM bleomycin pretreatment groups, respectively. These results indicate that bleomycin can promote the killing effect of antigen-specific T cells on tumor cells.

[0199] As shown in Figure 6, when B16F10 cells were co-cultured with OT-I T cells, the OT-I T cells did not secrete IFNγ. After B16F10 cells were pretreated with bleomycin and then co-cultured with OT-I T cells, the OT-I T cells still did not secrete IFNγ. However, when B16OVA cells were co-cultured with OT-I T cells, the OT-I T cells secreted IFNγ. After B16OVA cells were pretreated with bleomycin and then co-cultured with OT-I T cells, the IFNγ secretion level of the OT-I T cells significantly increased. These results indicate that bleomycin can promote the activation of antigen-specific T cells by tumor cells.

[0200] Example 4: BLM enhances the anti-tumor response of T cells

[0201] The clinical potential of bleomycin combined with adoptive T-cell therapy for tumor treatment was evaluated in a B16OVA cell mouse melanoma model. The specific method was as follows: On day 0 (D0), mice were subcutaneously inoculated with B16OVA cells (1×10⁻⁶ cells). 6 (Suspension of 100 μL per mL, diluted in pre-chilled PBS); on day 8 (D8), mouse tumors grew to 50-100 mm. 3 Mice were randomly divided into groups of 8 mice each, based on tumor volume and body weight. Starting from day 8, bleomycin (3 mg / kg) was injected intraperitoneally into the bleomycin group and the combination group (bleomycin + OT-IT), every two days. On day 11 (D11), 2 × 10⁻⁶ cells were reinfused into the OT-IT cell group (OT-IT) and the combination group via tail vein injection. 6 Preactivated OT-1 T cells (see also Figure 7A and the table below).

[0202] For mice used for anti-tumor efficacy analysis, mice were sacrificed at day 18 (D18), tumor weight was measured and mice were analyzed for tumor volume and body weight change, results are shown in FIG. 7B-D. As shown in FIG. 7B, no significant body weight loss was observed in mice of each group during the treatment. As shown in FIG. 7C and 7D, compared with the control group, both tumor volume and tumor weight of mice in the OT-I T cell group were significantly decreased, and both tumor volume and tumor weight of mice in the bleomycin group were slightly decreased but showed no statistical difference. Compared with the two individual treatment groups, both tumor volume and tumor weight of mice in the combination group were significantly decreased. The results indicated that bleomycin enhanced the anti-tumor response of T cells in vivo.

[0203] For mice used for Western blot, qPCR and immunofluorescence staining, mice were sacrificed at day 18 (D18), tumor tissues were removed, photographed and recorded, and then processed for analysis, results are shown in FIG. 8-10. As shown in FIG. 8, compared with the untreated group (OT-I T cell group), the content of B2m in tumor cells of the bleomycin treated group (combination group) was significantly increased, indicating that the expression level of MHC-I in tumor cells was significantly increased. As shown in FIG. 9, compared with the untreated group (OT-I T cell group), the expression levels of anti-tumor effector molecules GranzymeB (Gzmb), IFNy (Ifng) and perforin (Prfl) in the bleomycin treated group (combination group) were significantly increased. The results indicated that bleomycin could increase the expression of anti-tumor effector molecules in tumor tissues. As shown in FIG. 10, compared with the vehicle control, the level of GranzymeB in mice of the OT-I T cell group was higher, and the level of GranzymeB in mice of the bleomycin group was also increased. Most importantly, the expression level of GranzymeB in the tumor of mice in the combination group was significantly higher than that in mice of the OT-I T cell group. The results indicated that bleomycin could increase the expression level of GranzymeB in tumor tissues, and it synergistically promoted the expression level of GranzymeB in tumor tissues with the reinfusion of OT-I T cells.

[0204] For mice used for survival curve recording, mice were considered dead when any of the following occurred: (1) mice died; (2) the tumor of mice exceeded 2000 mm 3 ; (3) the tumor of mice showed severe ulceration; (4) the body weight of mice decreased by more than 10%. Survival analysis was performed at the end of the experiment, results are shown in FIG. 11. As shown in FIG. 11, the survival period of mice in the combination group was significantly longer than that of mice in the OT-I T cell group. The results indicated that bleomycin could increase the therapeutic effect of OT-I T cell reinfusion and prolong the survival period of mice.

[0205] For the mice used for immune cell infiltration analysis, the mice were sacrificed at day 14 (D14) and the tumor tissues were processed for analysis of tumor infiltrating immune cells in the tumor tissues by flow cytometry. The results are shown in Figure 12. As shown in Figure 12, the immune cell infiltration in the mice in the combination group was significantly more than that in the OT-I T cell group mice. The results indicated that bleomycin can increase the infiltration of immune cells in the tumor tissues.

[0206] Example 5 Bleomycin promotes the anti-tumor efficacy of T cell engager

[0207] The clinical potential of bleomycin in combination with T cell engager (BiTE) for treating tumors was further explored. There is a report of a bispecific antibody H2-scDb that targets CD3 on the surface of T cells on one end and p53 R175H / HLA-A*02:01 complex on the surface of tumor cell membrane. The effect of H2-scDb is closely related to the expression level of HLA-A on the surface of tumor cells. Bleomycin can increase the expression level of MHC-I on the surface of tumor cell membrane, so it was detected whether bleomycin can promote the activation of CD8 + T cells by H2-scDb.

[0208] After SK-BR-3 cells were treated with bleomycin, they were co-cultured with CD8+ T cells in the presence of H2-scDb antibody, and then the apoptosis of tumor cells was detected by crystal violet staining and the ability of CD8+ T cells to secrete IFNγ was detected by ELISA. The specific method is as follows: after SK-BR-3 cells were pretreated with 10 μM bleomycin, the medium containing bleomycin was removed, and the cells were washed with PBS, then a certain proportion (T cells: SK-BR-3 cells = 5: 1) of CD8+ T cells were added, and 0.3 nM of bispecific antibody H2-scDb was added, and after about 24 h of co-culture, the apoptosis of tumor cells was analyzed by crystal violet staining and CellTiter Glo Luminescent cell viability assay kit, and the ability of CD8 + T cells to secrete IFNγ was detected by ELISA using Mouse IFN-γ ELISA Set (BD Phar mingen). The results are shown in Figures 13A-C.

[0209] As shown in Figures 13A-C, due to the low expression level of MHC-I on the surface of SK-BR-3 cells, the presence of H2-scDb cannot promote the killing of tumor cells by CD8 + T cells, which is consistent with the results reported in the literature. In the absence of H2-scDb, SK-BR-3 cells pretreated with bleomycin can be killed by CD8 +Significant apoptosis occurred after T cell co-culture (Figures 13A and 13C), and CD8... + T cells secreted significantly increased IFNγ (Figure 13B). In the presence of H2-scDb, SK-BR-3 cells pretreated with bleomycin showed increased activity against CD8+. + T cell co-culture significantly increased apoptosis (Figures 13A and 13C), and CD8... + T cell secretion of IFNγ was significantly increased (Figure 13B). The results indicate that bleomycin can promote the inhibition of CD8 by H2-scDb. + Activation of T cells.

[0210] Example 6: Combination of bleomycin and anti-PD-L1 antibody enhances the anti-tumor effect of anti-PD-L1 antibody.

[0211] The clinical potential of bleomycin combined with immune checkpoint inhibitor therapy for tumor treatment was evaluated in a B16F10 cell mouse melanoma model. The specific method was as follows: On day 0 (D0), mice were subcutaneously inoculated with well-developed B16F10 cells (2 × 10⁻⁶ cells). 5 (Suspension of 100 μL per mL, diluted in pre-chilled PBS); on day 7 (D7), mouse tumors grew to 50-100 mm. 3 Mice were randomly divided into groups of 8 mice each, based on tumor volume and body weight. Starting on day 7, mice in the bleomycin group and the combination group (bleomycin + Anti-PD-L1) were intraperitoneally injected with bleomycin (3 mg / kg) every two days. On days 7 and 12, mice in the anti-PD-L1 antibody group (Anti-PD-L1) and the combination group (bleomycin + Anti-PD-L1) were intraperitoneally injected with 200 μg of anti-PD-L1 antibody (Anti-mouse PD-L1 (B7-H1), purchased from Bio X Cell, catalog number: BE0101) (see table below). On day 13 (D13), the mice were sacrificed, and tumor weight was measured. Changes in tumor volume and body weight were analyzed. The results are shown in Figures 14-16.

[0212] As shown in Figure 16, no significant decrease in body weight was observed in any group of mice during the treatment. As shown in Figures 14 and 15, compared to the control group, both the tumor volume and tumor weight decreased in the anti-PD-L1 antibody group and the bleomycin group. Compared to the two individual treatment groups, the tumor volume and tumor weight of the combination treatment group were significantly reduced. These results indicate that bleomycin can enhance the anti-tumor effect of the anti-PD-L1 antibody.

[0213] Example 7: Bleomycin enhances the killing effect of autologous tumor-infiltrating lymphocytes on primary bladder cancer cells by increasing MHC-I expression levels.

[0214] In this study, the clinical potential of bleomycin in combination with tumor infiltrating lymphocyte (TIL) therapy for treating tumors was further explored.

[0215] First, primary bladder cancer cell lines were successfully established from tumor tissues and urine samples of 8 bladder cancer patients. Four of these cell lines were derived from urine samples of the patients (BCC3, BCC16, BCC38 and BCC49), and the other four cell lines were derived from tumor samples of the patients (BCC1, BCC15, BCC101 and BCC102).

[0216] After treating the primary bladder cancer cell lines with different concentrations of bleomycin, the expression level of MHC-I molecules was analyzed by Western blotting, and the results are shown in Figure 17. As shown in Figure 17, the expression level of MHC-I in most of the primary bladder cancer cells was significantly increased after bleomycin treatment. The results showed that bleomycin can increase the expression level of MHC-I in primary bladder cancer cells.

[0217] Next, the toxic effect of bleomycin on primary bladder cancer cells was detected. After treating the primary bladder cancer cell lines with different concentrations of bleomycin for 72 h, the cell viability was detected by CellTiter-Glo Luminescent Cell Viability Assay Kit, and the results are shown in Figure 18. As shown in Figure 18, the IC50 (half maximal inhibitory concentration) value of bleomycin on most of the primary bladder cancer cells was more than 50 μΜ (BCC3, BCC38, BCC49, BCC1, BCC101 and BCC102), the IC50 of BCC16 was 10.44 μΜ, and the IC50 of BCC15 was 24.91 μΜ. The results showed that the toxicity of bleomycin on primary bladder cancer cells was very low.

[0218] Next, the ability of bleomycin to enhance the killing effect of autologous TILs on tumor cells was studied in tumor cell and TILs cell co-culture experiments. As shown in Figure 17, the primary bladder cancer cell line derived from BCC101 responded better to bleomycin treatment, while the primary bladder cancer cell line derived from BCC102 responded less to bleomycin treatment. Therefore, among the four tumor samples (BCC1, BCC15, BCC101 and BCC102), BCC101 and BCC102 were selected for the isolation and culture of tumor infiltrating lymphocytes. The cultured tumor infiltrating lymphocytes were analyzed by multicolor flow cytometry, and the results are shown in Figure 19. The results of Figure 19 showed that tumor infiltrating lymphocytes were successfully isolated and cultured from BCC101 and BCC102.

[0219] The primary bladder cancer cell lines derived from BCC101 and BCC102 were pretreated with bleomycin and then co-cultured with the corresponding autologous TILs cells, and the cell viability was detected by CellTiter Glo Luminescent Cell Viability Assay Kit. The results are shown in Figure 20. As shown in Figure 20, bleomycin pretreatment can significantly further enhance the killing effect of autologous tumor infiltrating lymphocytes on primary bladder cancer cells derived from BCC101, while the promotion of the killing effect of BCC102 autologous tumor infiltrating lymphocytes is not significant.

[0220] To further verify that bleomycin pretreatment can significantly further enhance the killing effect of autologous TILs on primary bladder cancer BCC101, after co-culture, the apoptosis of tumor cells was analyzed by crystal violet staining and flow cytometry. As shown in Figure 21, the primary bladder cancer cells derived from BCC101 were co-cultured with autologous TILs F1, F2 and F3, respectively, and the primary bladder cancer cells showed more obvious apoptosis, with an apoptosis rate of about 30%. The primary bladder cancer cells derived from BCC101 were pretreated with bleomycin and then co-cultured with autologous TILs F1, F2 and F3, and the apoptosis of the primary bladder cancer cells increased significantly, with an apoptosis rate of about 90%. The results show that bleomycin can enhance the killing effect of autologous tumor infiltrating lymphocytes on primary bladder cancer cells.

[0221] SEQUENCE LISTING

Claims

1. A pharmaceutical combination comprising bleomycin, a bleomycin analogue, or a combination thereof, and an immunotherapeutic agent involved in MHC-I and / or MHC-II dependent immune responses.

2. The pharmaceutical combination of claim 1, wherein the bleomycin is selected from the group consisting of BLM A2, BLM B2, BLM A5, and combinations thereof; more preferably, the bleomycin is a mixture of BLM A2 and BLM B2.

3. The pharmaceutical combination of claim 1, wherein the bleomycin analogue is selected from the group consisting of porfiromycin, rachelmycin, liblomycin, zorbamycin, corchomycin, and combinations thereof.

4. The pharmaceutical combination of any one of claims 1-3, wherein the immunotherapeutic agent targets an antigenic peptide-MHC-I complex (pMHC-I) and / or an antigenic peptide-MHC-II complex (pMHC-II), the antigenic peptide being selected from the group consisting of a tumor antigenic peptide, a viral antigenic peptide, a bacterial antigenic peptide, a fungal antigenic peptide, and a parasitic antigenic peptide.

5. The pharmaceutical combination of claim 5, wherein the antigenic peptide is a tumor antigenic peptide; preferably, the tumor antigenic peptide comprises an MHC-I restricted epitope selected from the group consisting of the following proteins: KRAS, MAGE1, gp100, hTERT, NY-ESO-1, hCGbeta, Her2 / Neu, Melan-A / MART-1, TARP, p53, p68, MIF, Proteinase 3 (PR1), WT1, HA-1, PRAME, CEA, MAGE-A3, and MAGE-A4; more preferably, the tumor antigenic peptide comprises an MHC-I restricted epitope selected from the group consisting of the following proteins: KRAS, gp100, and p53.

6. The pharmaceutical combination of any one of claims 1-5, wherein the immunotherapeutic agent is an anti-tumor immunotherapeutic agent; preferably, the anti-tumor immunotherapeutic agent is selected from the group consisting of an antibody, an antibody-drug conjugate, an immune cell, and combinations thereof.

7. The pharmaceutical combination of claim 6, wherein the anti-tumor immunotherapeutic agent is selected from the group consisting of cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), T cell receptor engineered T cells (TCR-Ts), and combinations thereof.

8. The pharmaceutical combination of claim 6, wherein the anti-tumor immunotherapeutic agent comprises a T cell receptor (TCR)-like antibody.

9. The pharmaceutical combination of claim 8, wherein the anti-tumor immunotherapeutic agent is selected from the group consisting of: a TCR-like antibody, a T cell engager, an immunotoxin, a chimeric antigen receptor T cell (CAR-T), and combinations thereof.

10. The pharmaceutical combination of claim 9, wherein the T cell engager is a T cell engaging bispecific antibody; preferably, the T cell engaging bispecific antibody is selected from the group consisting of antibody H2-scDb, antibody V2-scDb, and tebentafusp.

11. The pharmaceutical combination of any one of claims 1-3, wherein the immunotherapeutic agent is selected from the group consisting of an immune checkpoint inhibitor, a vaccine, and combinations thereof.

12. The pharmaceutical combination of claim 11, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-Ll antibody, an anti-PD-1 antibody, an anti- CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, and a combination thereof; preferably, the immune checkpoint inhibitor is an anti-PD-Ll antibody or an anti-PD-1 antibody.

13. The pharmaceutical combination of claim 11, wherein the vaccine is a tumor neoantigen-based vaccine.

14. A pharmaceutical composition comprising the pharmaceutical combination of any one of claims 1-13 and a pharmaceutically acceptable carrier.

15. A kit comprising the pharmaceutical combination of any one of claims 1-13 or the pharmaceutical composition of claim 14.

16. Use of the pharmaceutical combination of any one of claims 1-13, the pharmaceutical composition of claim 14, or the kit of claim 15 in the manufacture of a medicament for treating or preventing a cancer or an infectious disease.

17. The use of claim 16, wherein the cancer is a cancer in which cancer cells express low levels of MHC-I and / or MHC-II.

18. The use of claim 16 or 17, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, bone cancer, prostate cancer, lung cancer, adrenal cancer, bile duct cancer, bladder cancer, bronchus cancer, nervous tissue cancer, gallbladder cancer, stomach cancer, salivary gland cancer, esophagus cancer, small intestine cancer, cervix cancer, colon cancer, rectum cancer, liver cancer, ovarian cancer, pancreatic cancer, melanoma, pituitary adenoma, secretory adenoma, synovial sarcoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia. the infectious disease is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

19. Use of bleomycin or an analog thereof in the manufacture of an immune enhancer that enhances MHC-I and / or MHC-II dependent immune responses.

20. The use of claim 19, wherein the bleomycin is selected from the group consisting of BLM A2, BLM B2, BLM A5, and a combination thereof; more preferably, the bleomycin is a mixture of BLM A2 and BLM B2.

21. The use of claim 19, wherein the bleomycin analog is selected from the group consisting of porfiromycin, rachelmycin, liblomycin, zorbamycin, corchomycin, and a combination thereof.

22. The use of any one of claims 19-21, wherein the immune enhancer enhances the anti-tumor effect of an anti-tumor immunotherapeutic agent that participates in MHC-I and / or MHC-II dependent anti-tumor immune responses; preferably, the anti-tumor immunotherapeutic agent is an immunotherapeutic agent as defined in any one of claims 6-13.

23. The use of any one of claims 19-21, wherein the immune enhancer enhances an anti-infective immune response selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection. ​

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