Combining nonviable Streptococcus pyogenes cells with immune checkpoint inhibitors for the treatment of triple-negative breast cancer and non-muscle-invasive bladder cancer

Abstract

The present disclosure relates to a method for treating triple-negative breast cancer or non-muscle-invasive bladder cancer in a subject, comprising administering to the subject a composition comprising nonviable cells of Streptococcus pyogenes and an immune checkpoint inhibitor.

Description

[Background technology]

[0001] background Triple-negative breast cancer refers to breast tumors characterized by the absence of estrogen receptors, progesterone receptors, and HER2. Patients with triple-negative breast cancer do not respond to hormone therapy or trastuzumab-based treatment. Triple-negative breast cancer tends to be more aggressive, difficult to treat, and more likely to recur than other forms of the disease, such as hormone receptor-positive or HER2-positive breast cancer. Conventional chemotherapy is not very effective against triple-negative breast cancer, and novel treatment options are needed.

[0002] Bladder cancer is the 10th most common cancer worldwide, affecting approximately 500,000 people annually. Non-muscle-invasive bladder cancer (NMIBC) is defined as cancer confined to the bladder mucosa and submucosa and accounts for 75% of bladder cancer cases. The most common histological subtype is urothelial carcinoma. Non-muscle-invasive bladder cancer includes intramucosal papillary tumors (stage Ta), tumors invading the lamina propria (stage T1), and flat, high-grade lesions termed carcinoma in situ (CIS). NMIBC is primarily managed with local endoscopic / intravesical therapy and observation.

[0003] The risk of progression to muscle invasion or recurrence for non-muscle-invasive bladder cancer varies depending on the grade and depth of the tumor. For example, at one end of the spectrum, low-grade Ta bladder cancer recurs in approximately two-thirds of cases but rarely progresses to more invasive disease (only approximately 6%), whereas NMIBC with high-risk features, including high-grade T1, has a reported recurrence rate of approximately 50%. Furthermore, such cancers progress to muscle invasion in one in five patients, typically within two years of diagnosis.

[0004] BCG is the standard immunotherapy for NMIBC. Current treatment options after unsuccessful BCG therapy are limited to the standard of care, radical cystectomy. For patients who are unfit or unwilling to undergo cystectomy, the only two FDA-approved treatments for recurrent CIS are intravesical valrubicin and systemic pembrolizumab. Given the limited options in BCG-refractory disease, there is an unmet need for treatments for high-risk NMIBC, especially given the recent global BCG shortage and the high morbidity associated with radical cystectomy. Summary of the Invention

[0005] overview In one aspect, the present disclosure provides a method of treating triple-negative breast cancer in a subject, comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0006] In another aspect, the present disclosure provides a pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat triple-negative breast cancer, comprising nonviable cells of Streptococcus pyogenes.

[0007] In another aspect, the disclosure provides a medicament for use in combination with an immune checkpoint inhibitor to treat triple-negative breast cancer, including nonviable cells of Streptococcus pyogenes.

[0008] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD1 / PD-L1 / PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

[0009] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 / PD-L2 axis.

[0010] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0011] In some embodiments, the composition comprising nonviable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [Group A, Type 3] Su strain.

[0012] In some embodiments, the composition comprising nonviable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes.

[0013] In another aspect, the present disclosure provides a method of treating non-muscle-invasive bladder cancer in a subject, comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat non-muscle-invasive bladder cancer comprising nonviable cells of Streptococcus pyogenes.

[0015] In another aspect, the disclosure provides a medicament for use in combination with an immune checkpoint inhibitor to treat non-muscle invasive bladder cancer comprising nonviable cells of Streptococcus pyogenes.

[0016] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD1 / PD-L1 / PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

[0017] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 / PD-L2 axis.

[0018] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0019] In some embodiments, the composition comprising nonviable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [Group A, Type 3] Su strain.

[0020] In some embodiments, the composition comprising nonviable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes. [Brief explanation of the drawings]

[0021] [Figure 1] Assessment of EMT6 triple-negative breast cancer tumor growth (pre-randomization) in 54 mice enrolled in the study. [Figure 2] In vivo efficacy results of an exemplary composition comprising nonviable cells of Streptococcus pyogenes ("Composition 002" or "Comp. 002") in the EMT6 model. [Figure 3A-3B] In vivo efficacy results when Composition 002 was delivered intratumorally (FIG. 3A) or intravenously (FIG. 3B) as a single agent and in combination with an anti-PD1 antibody. [Figure 4A-4B] Weight measurement data for primary tumors (FIG. 4A) and spleens (FIG. 4B) from mice administered Composition 002 intratumorally as a single agent and in combination with an anti-PD1 antibody. [Figure 5A-5B] Gravimetric data of primary tumors (FIG. 5A) and spleens (FIG. 5B) from mice administered Composition 002 intravenously as a single agent and in combination with an anti-PD1 antibody. [Figures 6A-6B] Measurement of mean mouse body weight (FIG. 6A) and percent body weight change (FIG. 6B) in EMT6 tumor-bearing Balb / c mice (before randomization). [Figures 7A-7B] Measurement of mean mouse body weight (FIG. 7A) and percent body weight change (FIG. 7B) in EMT6 tumor-bearing Balb / c mice (after randomization, all groups). [Figure 8A-8B] Measurement of percent body weight change (after randomization) in EMT6 tumor-bearing Balb / c mice in the intratumoral delivery group (FIG. 8A) and the intravenous delivery group (FIG. 8B). [Figure 9A-9B] Analysis of % CD3+ T cells (gated on CD45+ CD3+) in the spleen (left) and tumor-TILs (right) of the intravenous (FIG. 9A) and intratumoral (FIG. 9B) treatment groups. [Figures 10A-10B] Analysis of CD4+ T cells (gated as CD45+ CD3+ CD8- CD4+) in the spleen (left) and tumor-TILs (right) of the intravenous (FIG. 10A) and intratumoral (FIG. 10B) treatment groups. [Figures 11A-11B] Analysis of CD8+ T cells (gated as CD45+ CD3+ CD4- CD8+) in the spleen (left) and tumor-TILs (right) of the intravenous (FIG. 11A) and intratumoral (FIG. 11B) treatment groups. [Figures 12A-12B] Analysis of NK+ cells (gated as CD45+ CD3- CD49b+-CD335+) in the spleen (left) and tumor-TILs (right) of the intravenous treatment group (FIG. 12A) and intratumoral treatment group (FIG. 12B). [Figures 13A-13B] Analysis of the proportion (percentage) of granulocytic MDSC cells (gated by CD45+ CD3- CD11b+-Ly6G+Ly6Clow) and monocytic MDSC in the spleen (left) and tumor-TIL (right) of the intravenous treatment group (Figure 13A) and intratumoral treatment group (Figure 13B). [Figures 14A-14B] Analysis of the ratio of granulocytic MDSC cells (gated by CD45+ CD3- CD11b+-Ly6G+Ly6Clow) to monocytic MDSC in the spleen (left) and tumor-TIL (right) of the intravenous treatment group (Figure 14A) and intratumoral treatment group (Figure 14B). [Figures 15A-15B] Analysis of regulatory T cells (gated by CD45+ CD3+ CD4+ CD25+ Fox3+) in the spleen (left) and tumor-TILs (right) of the intravenous (Figure 15A) and intratumoral (Figure 15B) treatment groups. [Figures 16A-16B] Analysis of TAMs (M1 / M2 ratio) gated as CD45+ CD3- F4 / 80+CD206- (M1) or CD45+ CD3- F4 / 80+CD206+ (M2) in the spleen (left) and tumor-TILs (right) of the intravenous treatment group (FIG. 16A) and intratumoral treatment group (FIG 16B). [Figures 17A-17B] Analysis of PD1+ / high T cells (gated as CD45+ CD3+) in the spleen (left) and tumor-TILs (right) of the intravenous (Figure 17A) and intratumoral (Figure 17B) treatment groups. [Figures 18A-18B] Analysis of PD1+ / high macrophages (gated as CD45+ CD3-F4 / 80+)+ in the spleen (left) and tumor-TILs (right) of the intravenous treatment group (Figure 18A) and intratumoral treatment group (Figure 18B). [Figures 19A-19B] Analysis of PD-L1+ / high T cells (gated as CD45+ CD3+) in the spleen (left) and tumor-TILs (right) of the intravenous treatment group (Figure 19A) and intratumoral treatment group (Figure 19B). [Figures 20A-20B]Analysis of PD-L1+ / high macrophages (gated by CD45+ CD3- F4 / 80+) in the spleen (left) and tumor-TILs (right) of the intravenous treatment group (Figure 20A) and intratumoral treatment group (Figure 20B). [Figures 21A-21K] Summary of flow cytometry analysis data for Composition 002 (dose 20 mg / Kg) in splenocytes and TILs in the intratumoral delivery group: CD3+ T cells (Figure 21A), CD4+ T cells (Figure 21B), CD8+ T cells (Figure 21C), NK cells (Figure 21D), MDSC / monocyte ratio (Figure 21E), regulatory T cells (Treg cells) (Figure 21F), macrophage M1 / M2 ratio (Figure 21G), PD1+ T cells (Figure 21H), PD-L1+ T cells (Figure 21I), PD-L1+ macrophages (Figure 21J), and PD-L1+ macrophages (Figure 21K). [Figures 22A-22B] Anti-tumor efficacy of Composition 002 and anti-mPD-1 in monotherapy and combination therapy in the MBT-2 tumor model. (Figure 22A) Mean absolute tumor volume ± SEM over time; (Figure 22B) Individual absolute tumor volumes at day 7 (the last day all groups remained on study). [Figures 23A-23F] Effect of Composition 002 and anti-mPD-1 treatment on leukocyte populations in MBT-2 tumors implanted subcutaneously (sc) in C3H mice - cell percentages. FC analysis of cells isolated from MBT-2 tumors from 10 mice per group at termination on day 10 (groups 1 and 8) or day 8 (groups 2-7). Cells were stained with antibody panels A and B as described in Example 2. The x-axis indicates the population of interest, displayed for each individual animal as a percentage of the parent population, indicated in red text below the x-axis label. The horizontal bar for each data set indicates the group mean value. (Figure 23A) Antibody panel A: CD45+ cells; (Figure 23B) Antibody panel A: CD4+ / CD8+ T cells and Treg cells; (Figure 23C) Antibody panel A: granulocyte and monocyte MDSC; (Figure 23D) Antibody panel B: CD45+CD3-CD11b+ cells; (Figure 23E) Antibody panel B: NK cells; (Figure 23F) Antibody panel B: M1 and M2 macrophages. [Figures 24A-24F]Effect of Composition 002 and anti-mPD-1 treatment on leukocyte populations—cell counts in MBT-2 tumors implanted subcutaneously (sc) in C3H mice. FC analysis of cells isolated from MBT-2 tumors from 10 mice per group at termination on day 10 (groups 1 and 8) or day 8 (groups 2-7). Cells were stained with antibody panels A and B shown below as described in Example 2. The x-axis indicates the population of interest, displayed for each individual animal as the number of cells within the population indicated by red text below the x-axis label. The horizontal bar for each data set indicates the group mean value. (Figure 24A) Antibody panel A: CD45+ cells; (Figure 24B) Antibody panel A: CD4+ / CD8+ T cells and Treg cells; (Figure 24C) Antibody panel A: granulocyte and monocyte MDSC; (Figure 24D) Antibody panel B: CD45+CD3-CD11b+ cells; (Figure 24E) Antibody panel B: NK cells; (Figure 24F) Antibody panel B: M1 and M2 macrophages. [Figure 25] Effect of treatment on mouse body weight. Group mean relative body weights over time for all groups are shown. [Figure 26] Composition 002 treatment does not alter CD4+ and CD8+ T cell counts. [Figures 27A-27B] Expression of immune checkpoint molecules in (FIG. 27A) CD4+ T cells and (FIG. 27B) CD8+ T cells (average of two donors) upon treatment with Composition 002. Data are presented as the mean (two donors / triplicates) ± SD. ** = P<0.01. *** = P<0.001. [Figures 28A-28D] Composition 002 treatment results in tumor apoptosis and the release of damage-associated molecular pattern molecules (DAMPs). MB49 cells were treated with different concentrations of Composition 002 for 24 hours. Mitoxantrone (1 uM) was used as a positive control. Figure 28A: The percentage of Annexin V-positive MB49 cells, a marker of apoptosis, was measured by flow cytometry. Data are presented as mean ± SEM. Figure 28B: Calreticulin-positive MB49 cells quantified by flow cytometry. Figure 28C. Extracellular ATP (eATP) luminescence was measured and calculated using the formula:

number

[0022] Detailed Description Before setting forth the present disclosure in more detail, it may be helpful to an understanding of the disclosure to provide definitions of certain terms used herein. Additional definitions are set forth throughout the disclosure.

[0023] Any concentration range, percentage range, ratio range, or integer range referred to herein, unless otherwise specified, is understood to include any integer value within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer) or subranges.

[0024] As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise specified.

[0025] As used herein, the terms "a," "an," and similar expressions are understood to refer to "one or more" of the listed components. The use of alternatives (e.g., "or") is understood to mean either one, both, or any combination thereof of the alternatives.

[0026] As used herein, the terms "comprise," "have," and "contain" are used interchangeably and the terms and variations thereof are intended to be construed as open ended.

[0027] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and the description includes instances where the element, component, event, or circumstance occurs and instances where it does not occur.

[0028] The term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody, such as an scFv, Fab, or Fab'2 fragment, that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody. Thus, the term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable region fragments (scFv), and single-domain antibodies (e.g., sdAb, sdFv, nanobodies). The term includes genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise specified, the term "antibody" is understood to include functional antibody fragments thereof. The term includes whole or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0029] As used herein, the term "immune checkpoint molecule" refers to one or more proteins, molecules, compounds, or complexes that provide inhibitory signals to help regulate or suppress immune responses. For example, immune checkpoint molecules include molecules that partially or completely block immune stimulation; molecules that reduce, prevent, or delay immune activation; or molecules that increase, activate, or upregulate immune suppression. As used herein, "regulating or suppressing an immune response" refers to reducing any one or more of antigen presentation, T cell activation, T cell proliferation, T cell effector function, cytokine secretion or production, and target cell lysis. Such regulation, control, or suppression may promote or enable the persistence of hyperproliferative diseases or disorders (e.g., cancer, chronic infection).

[0030] Exemplary immune checkpoint molecules include immune checkpoint ligands (e.g., PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9), immune checkpoint receptors (e.g., PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR), metabolic enzymes (e.g., arginase, indoleamine 2,3-dioxygenase (IDO)), immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), T regThe immune checkpoint molecules may include immune checkpoint molecules, immune cells, or any combination thereof. In certain embodiments, immune checkpoint molecules may induce immunosuppressive signals, for example, by modulating (e.g., inhibiting) antigen-specific T cell responses through ligand-receptor interactions. For example, T cells may express immune checkpoint receptors (e.g., PD-1, LAG3) on their surface, and antigen-presenting cells may express immune checkpoint receptor ligands (e.g., PD-L1, MHC / HLA molecules) on their surface. In further embodiments, the immune checkpoint molecule is a metabolic enzyme that inhibits immune responses through local depletion of amino acids essential for the survival and function of lymphocytes, particularly T cells. In yet further embodiments, the immune checkpoint molecule may be a signaling molecule, for example, an immunosuppressive cytokine (e.g., IL-10, IL-4, IL-1RA, IL-35).

[0031] Additionally, immune checkpoint molecules (e.g., IL-10) can cause a decrease in expression or levels of major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules, which in turn can reduce antigen presentation and thereby reduce, impede, or detectably interfere with T cell activation and the corresponding immune response.

[0032] "Immune checkpoint inhibitor" refers to any molecule that can directly or indirectly alter, interfere with, reduce, downregulate, block, suppress, abrogate, or degrade the expression, amount, or activity of an immune checkpoint molecule. Exemplary immune checkpoint inhibitors include small molecules, nucleic acid molecules (including vaccines, e.g., mRNA vaccines, and inhibitory nucleic acids, e.g., antisense oligonucleotides, siRNA, shRNA, and miRNA), peptides, proteins, antibodies or antigen-binding fragments thereof, fusion proteins, ribozymes, or gene editing systems.

[0033] As used herein, "triple-negative breast cancer" refers to a type of breast cancer that lacks expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2.

[0034] As used herein, "non-muscle-invasive bladder cancer" or "NMIBC" refers to a urothelial cancer that is confined to the bladder mucosa and submucosa and does not invade into or beyond the muscle layer.

[0035] The term "treat" or "treatment" as used herein refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a composition of the present disclosure to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" may also include treatment of animal models.

[0036] The term "treat" or "treatment" as used herein refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of a composition of the present disclosure to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" may also include treatment of animal models.

[0037] Compositions containing nonviable cells of Streptococcus pyogenes The present disclosure provides a composition comprising nonviable cells of Streptococcus pyogenes and an immune checkpoint inhibitor for use in combination with an immune checkpoint inhibitor to treat a subject with triple-negative breast cancer or non-muscle-invasive bladder cancer.

[0038] Streptococci are Gram-positive, catalase-negative, coagulase-negative cocci that occur in pairs or chains. They are classified into three groups based on the type of hemolysis they exhibit on blood agar: beta-hemolytic (complete lysis of red blood cells), hemolytic (green hemolysis), and gamma-hemolytic (no hemolysis). Beta-hemolytic streptococci are characterized as group A streptococci (Streptococcus pyogenes) and group B streptococci (Streptococcus agalactiae). Streptococcus pyogenes is a Gram-positive, non-spore-forming coccoid bacterium whose cells generally occur in chains or pairs. S. pyogenes is subdivided according to serotypes based on a large, highly variable cell surface antigen called the M protein (Lancefield, J. Exp. Med. 47, 9-10, 1928; Lancefield, J. Immunol. 89, 307-13, 1962). DNA sequencing of the gene encoding the M protein has become the most common method for determining S. pyogenes M types (emm sequence types). To date, 124 different M types have been identified (Facklam et al., Clin. Infect. Dis. 34, 28-38, 2002). M1, M28, M12, M3, M11, and M6 are some of the most common GAS types worldwide (Li et al., Infect. Dis. 188, 1587-92, 2003; O'Brien et al., Clin. Infect. Dis. 35, 268-76, 2002).

[0039] Any strain of S. pyogenes is contemplated for use within the disclosure of the present claims. In some embodiments of the present disclosure, the strain of S. pyogenes used is selected by the strain M protein (serotype). In some embodiments, the strain is an invasive strain. In some embodiments, the strain is isolated from a clinical sample. In some embodiments, the strain is a pathogenic strain. In some embodiments, the strain encodes an exotoxin. In some embodiments, the strain does not encode an exotoxin. In some embodiments, the strain is non-invasive. In some embodiments, the strain is non-pathogenic. In some embodiments, the strain is non-pathogenic due to a genetic mutation in a pathogenic strain.

[0040] In some embodiments, the composition comprises Streptococcus pyogenes M protein type 3.

[0041] In some embodiments, the composition comprises Streptococcus pyogenes (group A, type 3) Su strain.

[0042] In some embodiments of the present claimed disclosure, the strain of Streptococcus pyogenes used is selected from the group consisting of strains deposited with the American Type Culture Collection (ATCC). In some embodiments, the present disclosure provides a mixture comprising more than one strain of Streptococcus pyogenes. In some embodiments, the mixture comprises an Su strain and at least one additional Streptococcus pyogenes strain. Exemplary Streptococcus pyogenes strains, including Su strains, contemplated for use in the present disclosure are set forth in Table A. Further information regarding individual strains is provided at https: / / www.atcc.org / search#q=streptococcus%20pyogenes&sort=relevancy&numberOfResults=24&f:Productcategory=[Bacteria], which is incorporated herein by reference. In some embodiments, the composition comprises any one or more of the Streptococcus pyogenes strains identified in Table A below: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0043] In some embodiments, the composition comprising nonviable Streptococcus pyogenes is a pharmaceutical composition, and optionally comprises at least one pharmaceutically acceptable additive, such as a stabilizer, a buffer, a bulking agent, an antioxidant, a tonicity agent, an antibacterial agent, or any combination thereof.As known to those skilled in the art, in some cases, a component may have one or more activities, functions, or effects.For example, without wishing to be bound by theory, in some embodiments, some components (e.g., sodium chloride) may function as both a bulking agent and a tonicity agent.

[0044] In some embodiments, the composition is a lyophilized composition.

[0045] In some embodiments, the stabilizer is selected from magnesium hydroxide, calcium hydroxide, calcium carbonate, magnesium oxide, magnesium carbonate, magnesium sulfate, and combinations thereof.

[0046] In some embodiments, the stabilizer is magnesium hydroxide. In some embodiments, the stabilizer is calcium hydroxide. In some embodiments, the stabilizer is calcium carbonate. In some embodiments, the stabilizer is magnesium oxide. In some embodiments, the stabilizer is magnesium carbonate. In some embodiments, the stabilizer is magnesium sulfate.

[0047] In some embodiments, the stabilizer is present in the lyophilized composition at about 0.10% (w / w) to about 10.00% (w / w), about 0.10% (w / w) to about 5.00% (w / w), about 0.10% (w / w) to about 4.50% (w / w), about 0.10% (w / w) to about 4.00% (w / w), about 0.10% (w / w) to about 3.50% (w / w), It is present at about 0.10% (w / w) to about 3.00% (w / w), about 0.10% (w / w) to about 2.50% (w / w), about 0.10% (w / w) to about 2.00% (w / w), about 0.10% (w / w) to about 1.50% (w / w), about 0.10% (w / w) to about 1.00% (w / w), or about 0.10% (w / w) to about 0.50% (w / w).

[0048] In some embodiments, the stabilizer is present in the lyophilized composition at about 0.50% (w / w) to about 10.00% (w / w), about 0.50% (w / w) to about 5.00% (w / w), about 0.50% (w / w) to about 4.50% (w / w), about 0.50% (w / w) to about 4.00% (w / w), about 0.50% (w / w) to about 10 ... It is present at about 3.50% (w / w), about 0.50% (w / w) to about 3.00% (w / w), about 0.50% (w / w) to about 2.50% (w / w), about 0.50% (w / w) to about 2.00% (w / w), about 0.50% (w / w) to about 1.50% (w / w), or about 0.50% (w / w) to about 1.00% (w / w).

[0049] In some embodiments, the stabilizer is present in the lyophilized composition at about 1.00% (w / w) to about 10.00% (w / w), about 1.00% (w / w) to about 5.00% (w / w), about 1.00% (w / w) to about 4.50% (w / w), about 1.00% (w / w) to about 4.00% (w / w), about 1.00% (w / w) to about 3.50% (w / w), about 1.00% (w / w) to about 3.00% (w / w), about 1.00% (w / w) to about 2.50% (w / w), about 1.00% (w / w) to about 2.00% (w / w), or about 1.00% (w / w) to about 1.50% (w / w).

[0050] In some embodiments, the stabilizer is present in the lyophilized composition at about 1.50% (w / w) to about 10.00% (w / w), about 1.50% (w / w) to about 5.00% (w / w), about 1.50% (w / w) to about 4.50% (w / w), about 1.50% (w / w) to about 4.00% (w / w), about 1.50% (w / w) to about 3.50% (w / w), about 1.50% (w / w) to about 3.00% (w / w), about 1.50% (w / w) to about 2.50% (w / w), or about 1.50% (w / w) to about 2.00% (w / w).

[0051] In some embodiments, the stabilizer is present in the lyophilized composition at about 2.00% (w / w) to about 10.00% (w / w), about 2.00% (w / w) to about 5.00% (w / w), about 2.00% (w / w) to about 4.50% (w / w), about 2.00% (w / w) to about 4.00% (w / w), about 2.00% (w / w) to about 3.50% (w / w), about 2.00% (w / w) to about 3.00% (w / w), or about 2.00% (w / w) to about 2.50% (w / w).

[0052] In some embodiments, the stabilizer is present in the lyophilized composition at about 2.50% (w / w) to about 10.00% (w / w), about 2.50% (w / w) to about 5.00% (w / w), about 2.50% (w / w) to about 4.50% (w / w), about 2.50% (w / w) to about 4.00% (w / w), about 2.50% (w / w) to about 3.50% (w / w), or about 2.50% (w / w) to about 3.00% (w / w).

[0053] In some embodiments, the stabilizer is present in the lyophilized composition at about 3.00% (w / w) to about 10.00% (w / w), about 3.00% (w / w) to about 5.00% (w / w), about 3.00% (w / w) to about 4.50% (w / w), about 3.00% (w / w) to about 4.00% (w / w), or about 3.00% (w / w) to about 3.50% (w / w).

[0054] In some embodiments, the stabilizer is present in the lyophilized composition at about 3.50% (w / w) to about 10.00% (w / w), about 3.50% (w / w) to about 5.00% (w / w), about 3.50% (w / w) to about 4.50% (w / w), or about 3.50% (w / w) to about 4.00% (w / w).

[0055] In some embodiments, the stabilizer is present in the lyophilized composition at about 4.00% (w / w) to about 10.00% (w / w), about 4.00% (w / w) to about 5.00% (w / w), or about 4.00% (w / w) to about 4.50% (w / w).

[0056] In some embodiments, the stabilizer is present in the lyophilized composition at about 4.50% (w / w) to about 10.00% (w / w) or about 4.50% (w / w) to about 5.00% (w / w).

[0057] In some embodiments, the buffering agent is a phosphate salt. In some embodiments, the buffering agent is selected from potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, and sodium dihydrogen phosphate, and combinations thereof.

[0058] In some embodiments, the buffering agent is potassium dihydrogen phosphate. In some embodiments, the buffering agent is sodium phosphate.

[0059] In some embodiments, the buffering agent is present in the lyophilized composition at a concentration of about 1.00% (w / w) to about 25.00% (w / w), about 1.00% (w / w) to about 24.00% (w / w), about 1.00% (w / w) to about 23.00% (w / w), about 1.00% (w / w) to about 22.00% (w / w), about 1.00% (w / w) to about 21.00% (w / w), about 1. 00% (w / w) to about 20.00% (w / w), about 1.00% (w / w) to about 19.00% (w / w), about 1.00% (w / w) to about 18.00% (w / w), about 1.00% (w / w) to about 17.00% (w / w), about 1.00% (w / w) to about 16.00% (w / w), about 1.00% (w / w) to about 15.00% (w / w), about 1.00% (w / w) ) to about 14.00% (w / w), about 1.00% (w / w) to about 13.00% (w / w), about 1.00% (w / w) to about 12.00% (w / w), about 1.00% (w / w) to about 11.00% (w / w), about 1.00% (w / w) to about 10.00% (w / w), about 1.00% (w / w) to about 9.00% (w / w), about 1.00% (w / w) to about 8.00% (w / w), from about 1.00% (w / w) to about 7.00% (w / w), from about 1.00% (w / w) to about 6.00% (w / w), from about 1.00% (w / w) to about 5.00% (w / w), from about 1.00% (w / w) to about 4.00% (w / w), from about 1.00% (w / w) to about 3.00% (w / w), or from about 1.00% (w / w) to about 2.00% (w / w).

[0060] In some embodiments, the buffering agent is present in the lyophilized composition at about 5.00% (w / w) to about 25.00% (w / w), about 5.00% (w / w) to about 24.00% (w / w), about 5.00% (w / w) to about 23.00% (w / w), about 5.00% (w / w) to about 22.00% (w / w), about 5.0 0% (w / w) to approximately 21.00% (w / w), approximately 5.00% (w / w) to approximately 20.00% (w / w), approximately 5.00% (w / w) to approximately 19.00% (w / w), approximately 5.00% (w / w) to approximately 18.00% (w / w), approximately 5.00% (w / w) to approximately 17.00% (w / w), approximately 5.00% (w / w) Approximately 16.00% (w / w), approximately 5.00% (w / w) to approximately 15.00% (w / w), approximately 5.00% (w / w) to approximately 14.00% (w / w), approximately 5.00% (w / w) to approximately 13.00% (w / w), approximately 5.00% (w / w) to approximately 12.00% (w / w), approximately 5.00% (w / w) to approximately 11.00% ( and about 5.00% (w / w) to about 10.00% (w / w), about 5.00% (w / w) to about 9.00% (w / w), about 5.00% (w / w) to about 8.00% (w / w), about 5.00% (w / w) to about 7.00% (w / w), or 5.00% (w / w) to about 6.00% (w / w).

[0061] In some embodiments, the buffering agent is present in the lyophilized composition at a concentration of about 10.00% (w / w) to about 25.00% (w / w), about 10.00% (w / w) to about 24.00% (w / w), about 10.00% (w / w) to about 23.00% (w / w), about 10.00% (w / w) to about 22.00% (w / w), about 10.00% (w / w) to about 21.00% (w / w), about 10.00% (w / w) to about 20.00% (w / w), about 10.00% (w / w) to about 19.00% (w / w), about 10.00% (w / w) to about 18.00% (w / w), or about 10.00% (w / w) to about 17.00% (w / w). 0.00% (w / w), about 10.00% (w / w) to about 16.00% (w / w), about 10.00% (w / w) to about 15.00% (w / w), about 10.00% (w / w) to about 14.00% (w / w), about 10.00% (w / w) to about 13.00% (w / w), about 10.00% (w / w) to about 12.00% (w / w), about 10.00% (w / w) to about 11.00% (w / w).

[0062] In some embodiments, the buffering agent is present in the lyophilized composition at about 15.00% (w / w) to about 25.00% (w / w), about 15.00% (w / w) to about 24.00% (w / w), about 15.00% (w / w) to about 23.00% (w / w), about 15.00% (w / w) to about 22.00% (w / w), about 15.00% (w / w) to about 2 It is present at 1.00% (w / w), about 15.00% (w / w) to about 20.00% (w / w), about 15.00% (w / w) to about 19.00% (w / w), about 15.00% (w / w) to about 18.00% (w / w), about 15.00% (w / w) to about 17.00% (w / w), or about 15.00% (w / w) to about 16.00% (w / w).

[0063] In some embodiments, the buffering agent is present in the lyophilized composition at about 18.00% (w / w) to about 25.00% (w / w), about 18.00% (w / w) to about 24.00% (w / w), about 18.00% (w / w) to about 23.00% (w / w), about 18.00% (w / w) to about 22.00% (w / w), about 18.00% (w / w) to about 21.00% (w / w), about 18.00% (w / w) to about 20.00% (w / w), or about 18.00% (w / w) to about 19.00% (w / w).

[0064] In some embodiments, the bulking agent is selected from sodium chloride, mannitol, sucrose, lactose, dextran, trehalose, glycine, maltose, and combinations thereof.

[0065] In some embodiments, the bulking agent is sodium chloride. In some embodiments, the bulking agent is mannitol. In some embodiments, the bulking agent is sucrose. In some embodiments, the bulking agent is lactose. In some embodiments, the bulking agent is dextran. In some embodiments, the bulking agent is trehalose. In some embodiments, the bulking agent is maltose. In some embodiments, the bulking agent is glycine.

[0066] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.05% (w / w) to about 3.00% (w / w), about 0.05% (w / w) to about 2.50% (w / w), about 0.05% (w / w) to about 2.00% (w / w), about 0.05% (w / w) to about 1.50% (w / w), about 0.05% (w / w) to about 1.25% (w / w), about 0.05% (w / w) to about 1.00% (w / w), about 0.05% (w / w) to about 0.75% (w / w), about 0.05% (w / w) to about 0.50% (w / w), or about 0.05% (w / w) to about 0.25% (w / w).

[0067] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.25% (w / w) to about 3.00% (w / w), about 0.25% (w / w) to about 2.50% (w / w), about 0.25% (w / w) to about 2.00% (w / w), about 0.25% (w / w) to about 1.50% (w / w), about 0.25% (w / w) to about 1.25% (w / w), about 0.25% (w / w) to about 1.00% (w / w), about 0.25% (w / w) to about 0.75% (w / w), or about 0.25% (w / w) to about 0.50% (w / w).

[0068] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.50% (w / w) to about 3.00% (w / w), about 0.50% (w / w) to about 2.50% (w / w), about 0.50% (w / w) to about 2.00% (w / w), about 0.50% (w / w) to about 1.50% (w / w), about 0.50% (w / w) to about 1.25% (w / w), about 0.50% (w / w) to about 1.00% (w / w), or about 0.50% (w / w) to about 0.75% (w / w).

[0069] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.75% (w / w) to about 3.00% (w / w), about 0.75% (w / w) to about 2.50% (w / w), about 0.75% (w / w) to about 2.00% (w / w), about 0.75% (w / w) to about 1.50% (w / w), about 0.75% (w / w) to about 1.25% (w / w), or about 0.75% (w / w) to about 1.00% (w / w).

[0070] In some embodiments, the bulking agent is present in the lyophilized composition at about 1.00% (w / w) to about 3.00% (w / w), about 1.00% (w / w) to about 2.50% (w / w), about 1.00% (w / w) to about 2.00% (w / w), about 1.00% (w / w) to about 1.50% (w / w), or about 1.00% (w / w) to about 1.25% (w / w).

[0071] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.80% (w / w) to about 1.40% (w / w).

[0072] In some embodiments, the tonicity adjusting agent is selected from mannitol, D-mannitol, trehalose, αα-trehalose dihydrate, sucrose, dextrose, sodium chloride, and maltose. In some embodiments, the tonicity adjusting agent is mannitol. In some embodiments, the tonicity adjusting agent is D-mannitol. In some embodiments, the tonicity adjusting agent is trehalose. In some embodiments, the tonicity adjusting agent is αα-trehalose dihydrate. In another embodiment, the tonicity adjusting agent is sucrose. In another embodiment, the tonicity adjusting agent is dextrose. In another embodiment, the tonicity adjusting agent is sodium chloride. In some embodiments, the tonicity adjusting agent is maltose.

[0073] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.05% (w / w) to about 2.00% (w / w), about 0.05% (w / w) to about 1.50% (w / w), about 0.05% (w / w) to about 1.25% (w / w), about 0.05% (w / w) to about 1.00% (w / w), about 0.05% (w / w) to about 0.75% (w / w), about 0.05% (w / w) to about 0.50% (w / w), or about 0.05% (w / w) to about 0.25% (w / w).

[0074] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.25% (w / w) to about 2.00% (w / w), about 0.25% (w / w) to about 1.50% (w / w), about 0.25% (w / w) to about 1.25% (w / w), about 0.25% (w / w) to about 1.00% (w / w), about 0.25% (w / w) to about 0.75% (w / w), or about 0.25% (w / w) to about 0.50% (w / w).

[0075] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.50% (w / w) to about 2.00% (w / w), about 0.50% (w / w) to about 1.50% (w / w), about 0.50% (w / w) to about 1.25% (w / w), about 0.50% (w / w) to about 1.00% (w / w), or about 0.50% (w / w) to about 0.75% (w / w).

[0076] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.75% (w / w) to about 2.00% (w / w), about 0.75% (w / w) to about 1.50% (w / w), about 0.75% (w / w) to about 1.25% (w / w), or about 0.75% (w / w) to about 1.00% (w / w).

[0077] In some embodiments, the tonicity agent is present in the lyophilized composition at about 1.00% (w / w) to about 2.00% (w / w), about 1.00% (w / w) to about 1.50% (w / w), or about 1.00% (w / w) to about 1.25% (w / w).

[0078] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.05% (w / w) to about 10.00% (w / w), about 0.05% (w / w) to about 6.00% (w / w), about 0.05% (w / w) to about 5.50% (w / w), about 0.05% (w / w) to about 5.00% (w / w), about 0.05% (w / w) to about 4.50% (w / w), or about 0.05% (w / w) to about 4.00% (w / w).

[0079] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.25% (w / w) to about 10.00% (w / w), about 0.25% (w / w) to about 6.00% (w / w), about 0.25% (w / w) to about 5.50% (w / w), about 0.25% (w / w) to about 5.00% (w / w), about 0.25% (w / w) to about 4.50% (w / w), or about 0.25% (w / w) to about 4.00% (w / w).

[0080] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.50% (w / w) to about 10.00% (w / w), about 0.50% (w / w) to about 6.00% (w / w), about 0.50% (w / w) to about 5.50% (w / w), about 0.50% (w / w) to about 5.00% (w / w), about 0.50% (w / w) to about 4.50% (w / w), or about 0.50% (w / w) to about 4.00% (w / w).

[0081] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.75% (w / w) to about 10.00% (w / w), about 0.75% (w / w) to about 6.00% (w / w), about 0.75% (w / w) to about 5.50% (w / w), about 0.75% (w / w) to about 5.00% (w / w), about 0.75% (w / w) to about 4.50% (w / w), or about 0.75% (w / w) to about 4.00% (w / w).

[0082] In some embodiments, the tonicity agent is present in the lyophilized composition at about 1.00% (w / w) to about 10.00% (w / w), about 1.00% (w / w) to about 6.00% (w / w), about 1.00% (w / w) to about 5.50% (w / w), about 1.00% (w / w) to about 5.00% (w / w), about 1.00% (w / w) to about 4.50% (w / w), or about 1.00% (w / w) to about 4.00% (w / w).

[0083] In some embodiments, the tonicity agent is present in the lyophilized composition at about 0.80% (w / w) to about 1.40% (w / w).

[0084] In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce an osmolality equivalent to about 0.25% saline (w / w) to about 3% saline (w / w) when the lyophilized composition is reconstituted with water. In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce an osmolality equivalent to about 1% saline (w / w) to about 2% saline (w / w) when the lyophilized composition is reconstituted with water. In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce a hyperosmotic solution. In some embodiments, the hyperosmotic solution has an osmolality higher than 0.9% saline (w / w).

[0085] In some embodiments, the antioxidant is selected from methionine, cysteine, histidine, arginine, lysine, and combinations thereof.

[0086] In some embodiments, the antioxidant is methionine. In some embodiments, the antioxidant is L-methionine. In some embodiments, the antioxidant is cysteine. In some embodiments, the antioxidant is L-cysteine. In some embodiments, the antioxidant is histidine. In some embodiments, the antioxidant is L-histidine. In some embodiments, the antioxidant is arginine. In some embodiments, the antioxidant is L-arginine. In some embodiments, the antioxidant is lysine. In some embodiments, the antioxidant is L-lysine.

[0087] In some embodiments, the antioxidant is present in the lyophilized composition at about 0.10% (w / w) to about 15.00% (w / w), about 0.10% (w / w) to about 10.00% (w / w), about 0.10% (w / w) to about 7.50% (w / w), about 0.10% (w / w) to about 5.00% (w / w), about 0.10% (w / w) to about 4.50% (w / w), about 0.10% (w / w) to about 4.00% (w / w), It is present at about 0.10% (w / w) to about 3.50% (w / w), about 0.10% (w / w) to about 3.00% (w / w), about 0.10% (w / w) to about 2.50% (w / w), about 0.10% (w / w) to about 2.00% (w / w), about 0.10% (w / w) to about 1.50% (w / w), about 0.10% (w / w) to about 1.00% (w / w), or about 0.10% (w / w) to about 0.50% (w / w).

[0088] In some embodiments, the antioxidant is present in the lyophilized composition at about 2.50% (w / w) to about 15.00% (w / w), about 2.50% (w / w) to about 10.00% (w / w), 2.50% (w / w) to about 7.50% (w / w), about 2.50% (w / w) to about 5.00% (w / w), about 2.50% (w / w) to about 4.50% (w / w), about 2.50% (w / w) to about 4.00% (w / w), about 2.50% (w / w) to about 3.50% (w / w), or about 2.50% (w / w) to about 3.00% (w / w).

[0089] In some embodiments, the antioxidant is present in the lyophilized composition at about 5.00% (w / w) to about 15.00% (w / w), about 5.00% (w / w) to about 10.00% (w / w), or about 5.00% (w / w) to about 7.50% (w / w).

[0090] In some embodiments, the antioxidant is present in the lyophilized composition at about 7.50% (w / w) to about 15.00% (w / w) or about 7.50% (w / w) to about 10.00% (w / w).

[0091] In some embodiments, the antioxidant is present in the lyophilized composition at about 6.00% (w / w) to about 8.00% (w / w).

[0092] In some embodiments, the antibacterial agent is penicillin. In some embodiments, the antibacterial agent is selected from penicillin G or a pharmaceutically acceptable salt thereof, penicillin V or a pharmaceutically acceptable salt thereof, and combinations thereof.

[0093] In some embodiments, the antibacterial agent is selected from penicillin G potassium (benzylpenicillin), penicillin V potassium (penicillin VK), and combinations thereof.

[0094] In some embodiments, the antibacterial agent is penicillin G potassium (benzylpenicillin). In some embodiments, the antibacterial agent is penicillin V.

[0095] In some embodiments, the antimicrobial agent is present in the lyophilized composition at about 1.00% (w / w) to about 85.00% (w / w), about 1.00% (w / w) to about 75.00% (w / w), about 1.00% (w / w) to about 65.00% (w / w), about 1.00% (w / w) to about 55.00% (w / w), about 1.00% (w / w) to about 45.00% (w / w), about 1.00% (w / w) to about 35.00% (w / w), about 1.00% (w / w) to about 25.00% (w / w), about 1.00% (w / w) to about 15.00% (w / w), or about 1.00% (w / w) to about 5.00% (w / w).

[0096] In some embodiments, the antimicrobial agent is present in the lyophilized composition at about 15.00% (w / w) to about 85.00% (w / w), about 15.00% (w / w) to about 75.00% (w / w), about 15.00% (w / w) to about 65.00% (w / w), about 15.00% (w / w) to about 55.00% (w / w), about 15.00% (w / w) to about 45.00% (w / w), about 15.00% (w / w) to about 35.00% (w / w), or about 15.00% (w / w) to about 25.00% (w / w).

[0097] In some embodiments, the antimicrobial agent is present in the lyophilized composition at about 30.00% (w / w) to about 85.00% (w / w), about 30.00% (w / w) to about 75.00% (w / w), about 30.00% (w / w) to about 65.00% (w / w), about 30.00% (w / w) to about 55.00% (w / w), about 30.00% (w / w) to about 45.00% (w / w), or about 30.00% (w / w) to about 35.00% (w / w).

[0098] In some embodiments, the antimicrobial agent is present in the lyophilized composition at about 55.00% (w / w) to about 85.00% (w / w), about 55.00% (w / w) to about 75.00% (w / w), or about 55.00% (w / w) to about 65.00% (w / w).

[0099] In some embodiments, the antimicrobial agent is present in the lyophilized composition at about 60.00% (w / w) to about 85.00% (w / w), about 60.00% (w / w) to about 75.00% (w / w), or about 60.00% (w / w) to about 65.00% (w / w).

[0100] In some embodiments, Streptococcus pyogenes is treated with benzylpenicillin. In some embodiments, Streptococcus pyogenes is treated with benzylpenicillin and hydrogen peroxide. In some embodiments, Streptococcus pyogenes is heated after benzylpenicillin treatment. For example, benzylpenicillin-treated Streptococcus pyogenes can be incubated at 30-38°C for more than 10 minutes, preferably 10-45 minutes, and then further heated at 38-50°C for 20-60 minutes. An exemplary method for producing a composition of the present disclosure includes starting with a main culture of S. pyogenes; harvesting S. pyogenes cells from the main culture; treating the S. pyogenes cells with hydrogen peroxide; washing the S. pyogenes cells; resuspending the S. pyogenes cells in a suspension culture; treating the S. pyogenes cells with benzylpenicillin; heating the S. pyogenes cells; preparing a final suspension of S. pyogenes cells; and lyophilizing the S. pyogenes cells.

[0101] In some embodiments, the freeze-dried composition comprising nonviable Streptococcus pyogenes comprises nonviable Streptococcus pyogenes e (group A, type 3) strain Su, maltose, magnesium sulfate, potassium dihydrogen phosphate, 0.9% sodium chloride, methionine, and benzylpenicillin.

[0102] Exemplary quantitative formulations for various suggested dose strengths of exemplary compositions containing nonviable Streptococcus pyogenes are shown in Table 13A. These compositions are based on lyophilized products. Exemplary quantitative formulations for exemplary compositions suspended in 0.9% saline are shown in Table 13B.

[0103] In some embodiments, the composition comprising nonviable S. pyogenes comprises intact S. pyogenes cells.

[0104] In some embodiments, viable S. pyogenes is determined by testing for bacterial growth. In a preferred embodiment, viable S. pyogenes is detected using a growth test for hemolytic streptococci using blood agar.

[0105] In some embodiments, the composition contains no detectable viable Streptococcus pyogenes after storage for an extended period of time (e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months) under various temperatures (e.g., about -15°C to about 40°C, about -5°C to about 30°C, about 0°C to about 20°C, about 0°C to about 10°C) and humidities (e.g., about 40% RH, about 45% RH, about 50% RH, about 55% RH, about 60% RH, about 65% RH, about 70% RH, about 75% RH, about 80% RH, about 85% RH, or about 90% RH).

[0106] In some embodiments, the composition contains no detectable viable Streptococcus pyogenes after storage below 10°C and above 0°C for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 20 months, about 22 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months.

[0107] As used herein, a composition of the present disclosure is considered to have "retained potency" if, after a specified period of time, the measured potency of the composition (as determined by common methods known in the art) is within a predetermined range of the potency of an appropriate standard. In some embodiments, potency is calculated using a cytokine release assay. In some embodiments, the range is 20-180% of the standard. In some embodiments, the range is 40-160% of the standard. In some embodiments, the range is 50-150% of the standard. In some embodiments, the range is 60-140% of the standard. In some embodiments, the range is 70-130% of the standard. In some embodiments, the range is 80-120% of the standard. In a preferred embodiment, the range is 60-140% of the standard.

[0108] In some embodiments, the compositions retain potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the compositions described herein.

[0109] In some embodiments, the composition retains potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the composition at the conditions described herein of about 2°C to about 8°C.

[0110] In some embodiments, the composition retains potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the composition at about 23°C to about 27°C and about 55% to about 65% relative humidity as described herein.

[0111] In some embodiments, the compositions described herein remain viable for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage. Thus, the potency of nonviable S. pyogenes cells and / or the relative percentage amount of nonviable S. pyogenes cells in the composition is stable, i.e., does not change or does not change by more than about 25%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0112] In some embodiments, the composition comprising nonviable S. pyogenes is a lyophilized composition or formulation, hi some embodiments, the composition comprising nonviable S. pyogenes is a lyophilized powder.

[0113] In some embodiments, the composition comprising nonviable Streptococcus pyogenes is OK-432 (Picibanil). TM OK-432 is a dried, lyophilized biological product manufactured by treating the Su strain of Streptococcus pyogenes (group A, type 3) with benzylpenicillin and heating it. OK-432 does not undergo further processing, such as isolation, extraction, or purification. The bacterial cells remain intact. However, when administered to humans, they lose their ability to grow and do not cause streptococcal infection.

[0114] In some embodiments, compositions comprising nonviable S. pyogenes were developed from the same genetically distinct Group A, Type 3, S. pyogenes master cell bank as OK-432.

[0115] Methods for producing compositions containing nonviable Streptococcus pyogenes are described, for example, in U.S. Pat. No. 3,477,914; U.S. Pat. No. 3,632,746; Aoki et al., J. Natl. Cancer Inst. 56:687 (1976), each of which is incorporated herein by reference in its entirety.

[0116] In some embodiments, the lyophilized compositions described herein can be further used to produce a liquid composition of Streptococcus pyogenes. In some embodiments, the liquid composition is a suspension.

[0117] In some embodiments, the liquid composition comprises a mixture of a lyophilized composition described herein and water. In some embodiments, the liquid composition comprises a mixture of a lyophilized composition described herein and an aqueous NaCl solution. In some embodiments, the concentration of sodium chloride is about 0.5% to about 1.5%, about 0.6% to about 1.4%, about 0.7% to about 1.3%, about 0.8% to about 1.2%, or about 0.8% to about 1.0% (w / v). In some embodiments, the concentration of sodium chloride is about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5% (w / v). In some embodiments, the solvent for the lyophilized composition is a 0.9% sodium chloride solution.

[0118] In some embodiments, the lyophilized composition is suspended in a liquid (eg, an isotonic sodium chloride solution) to prepare a suspension at a concentration of about 0.005 mg / mL to about 0.01 mg / mL.

[0119] In some embodiments, Klinische Einheit (KE) is used as a unit of measure for a dose of a composition containing nonviable cells of Streptococcus pyogenes. 1 KE is approximately 1 x 10 8 Equivalent to 0.1 mg of lyophilized streptococci containing 100 cells.

[0120] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 15 KE to about 200 KE, about 15 KE to about 150 KE, about 15 KE to about 100 KE, about 15 KE to about 90 KE, about 15 KE to about 80 KE, about 15 KE to about 70 KE, about 15 KE to about 60 KE, about 15 KE to about 50 KE, about 15 KE to about 40 KE, about 15 KE to about 30 KE, or about 15 KE to about 20 KE.

[0121] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 20 KE to about 200 KE, about 20 KE to about 150 KE, about 20 KE to about 100 KE, about 20 KE to about 90 KE, about 20 KE to about 80 KE, about 20 KE to about 70 KE, about 20 KE to about 60 KE, about 20 KE to about 50 KE, about 20 KE to about 40 KE, or about 20 KE to about 30 KE.

[0122] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 30 KE to about 200 KE, about 30 KE to about 150 KE, about 30 KE to about 100 KE, about 30 KE to about 90 KE, about 30 KE to about 80 KE, about 30 KE to about 70 KE, about 30 KE to about 60 KE, about 30 KE to about 50 KE, or about 30 KE to about 40 KE.

[0123] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 40 KE to about 200 KE, about 40 KE to about 150 KE, about 40 KE to about 100 KE, about 40 KE to about 90 KE, about 40 KE to about 80 KE, about 40 KE to about 70 KE, about 40 KE to about 60 KE, or about 40 KE to about 50 KE.

[0124] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 50 KE to about 200 KE, about 50 KE to about 150 KE, about 50 KE to about 100 KE, about 50 KE to about 90 KE, about 50 KE to about 80 KE, about 50 KE to about 70 KE, or about 50 KE to about 60 KE.

[0125] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of at least 15 KE, at least 20 KE, at least 30 KE, at least 40 KE, at least 50 KE, at least 60 KE, at least 70 KE, at least 80 KE, at least 90 KE, at least 100 KE, or at least 150 KE.

[0126] In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, about 60 KE, about 70 KE, about 80 KE, about 90 KE, about 100 KE, or about 150 KE. In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 15 ±1, ±2, ±3, or ±4 KE, 20 ±1, ±2, ±3, or ±4 KE, 30 ±1, ±2, ±3, or ±4 KE, 40 ±1, ±2, ±3, or ±4 KE, 50 ±1, ±2, ±3, or ±4 KE, 60 ±1, ±2, ±3, or ±4 KE, 70 ±1, ±2, ±3, or ±4 KE, 80 ±1, ±2, ±3, or ±4 KE, 90 ±1, ±2, ±3, or ±4 KE, 100 ±1, ±2, ±3, or ±4 KE, or 150 ±1, ±2, ±3, or ±4 KE.

[0127] In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, or about 60 KE. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 15 ±1, ±2, ±3, or ±4 KE, 20 ±1, ±2, ±3, or ±4 KE, 30 ±1, ±2, ±3, or ±4 KE, 40 ±1, ±2, ±3, or ±4 KE, 50 ±1, ±2, ±3, or ±4 KE, or 60 ±1, ±2, ±3, or ±4 KE.

[0128] In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of about 15 KE, about 20 KE, about 30 KE, or about 40 KE. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 15 ±1, ±2, ±3, or ±4 KE, 20 ±1, ±2, ±3, or ±4 KE, 30 ±1, ±2, ±3, or ±4 KE, or 40 ±1, ±2, ±3, or ±4 KE.

[0129] In some embodiments, the nonviable cells of Streptococcus pyogenes comprise at least 3.5% (w / w), at least 3.6% (w / w), at least 3.7% (w / w), at least 3.8% (w / w), at least 3.9% (w / w), at least 4.0% (w / w), at least 4.1% (w / w), at least 4.2% (w / w), at least 4.3% (w / w), at least 4.4% (w / w), at least 4.5% (w / w), at least 4.6% (w / w), at least is also present in the lyophilized composition in an amount of 4.7% (w / w), at least 4.8% (w / w), at least 4.9% (w / w), at least 5.0% (w / w), at least 5.5% (w / w), at least 6.0% (w / w), at least 6.5% (w / w), at least 7.0% (w / w), at least 7.5% (w / w), at least 8.0% (w / w), at least 8.5% (w / w), at least 9.0% (w / w), at least 9.5% (w / w), or at least 10.0% (w / w). In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of at least 3.5% (w / w), at least 3.6% (w / w), at least 3.7% (w / w), at least 3.8% (w / w), at least 3.9% (w / w), at least 4.0% (w / w), at least 4.1% (w / w), at least 4.2% (w / w), at least 4.3% (w / w), at least 4.4% (w / w), at least 4.5% (w / w), at least 4.6% (w / w), at least 4.7% (w / w), at least 4.8% (w / w), at least 4.9% (w / w), or at least 5.0% (w / w) of the total weight of the lyophilized composition.

[0130] In some embodiments, the nonviable cells of Streptococcus pyogenes are about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), about 5.0% (w / w), about 5.1% (w / w), about 5.2% (w / w), about 5.3% (w / w), about 5.4% (w / w), about 5.5% (w / w), about 5.6% (w / w), about 5.7% (w / w), about 5.8% (w / w), about 5.9% (w / w), about 6.0% (w / w), about 6.1% (w / w), about 6.2% (w / w), about 6.3% (w / w), about 6.4% (w / w), about 6.5% (w / w), about 6.6% (w / w), about 6.7% (w / w), about 6.8% (w / w), about 6.9% (w / w), about 7.0% (w / w), about 7.1% (w / w), about 7.2% (w / w), about 7.3% (w / w), about 7.4% (w / w), about 7.5% (w / w), about 7.6% (w / w), about 7.7% (w / w), about 7.8% (w / w), about 7.9% It is present in the lyophilized composition in an amount of about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), about 5.0% (w / w), about 5.5% (w / w), about 6.0% (w / w), about 6.5% (w / w), about 7.0% (w / w), about 7.5% (w / w), about 8.0% (w / w), about 8.5% (w / w), about 9.0% (w / w), about 9.5% (w / w), or about 10.0% (w / w). In some embodiments, the nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition.

[0131] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 15 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition.

[0132] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 20 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 20±1,±2,±3, or ±4 KE and in an amount of about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), or about 4.1% (w / w) of the total weight of the lyophilized composition. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 40±1,±2,±3, or ±4 KE and in an amount of about 3.9% (w / w) of the total weight of the lyophilized composition.

[0133] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 30 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition.

[0134] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 40 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 40 ± 1, ± 2, ± 3, or ± 4 KE and in an amount of about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), or about 4.1% (w / w) of the total weight of the lyophilized composition. In some embodiments, the nonviable cells of S. pyogenes are present in the lyophilized composition in an amount of 40 ± 1, ± 2, ± 3, or ± 4 KE and in an amount of about 3.9% (w / w) of the total weight of the lyophilized composition.

[0135] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition at 50 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition.

[0136] In some embodiments, nonviable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 60 ±1, ±2, ±3, or ±4 KE and in an amount of about 3.5% (w / w), about 3.6% (w / w), about 3.7% (w / w), about 3.8% (w / w), about 3.9% (w / w), about 4.0% (w / w), about 4.1% (w / w), about 4.2% (w / w), about 4.3% (w / w), about 4.4% (w / w), about 4.5% (w / w), about 4.6% (w / w), about 4.7% (w / w), about 4.8% (w / w), about 4.9% (w / w), or about 5.0% (w / w) of the total weight of the lyophilized composition.

[0137] In some embodiments, the relative percentage of nonviable S. pyogenes cells increases when the amount of nonviable S. pyogenes cells in the lyophilized composition increases, e.g., the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE, 30 KE, 20 KE, 15 KE, or 10 KE.

[0138] In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE, 30 KE, 20 KE, or 15 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20 ±3, ±2, or ±1%, 25 ±3, ±2, or ±1%, 30 ±3, ±2, or ±1%, 35 ±3, ±2, or ±1%, 40 ±3, ±2, or ±1%, 45 ±3, ±2, or ±1%, or 50 ±3, ±2, or ±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE, 30 KE, 20 KE, or 15 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE, 30 KE, 20 KE, or 15 KE than when they are present in an amount of 10 KE.

[0139] In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 15 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20 ±3, ±2, or ±1%, 25 ±3, ±2, or ±1%, 30 ±3, ±2, or ±1%, 35 ±3, ±2, or ±1%, 40 ±3, ±2, or ±1%, 45 ±3, ±2, or ±1%, or 50 ±3, ±2, or ±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 15 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 15 KE than when they are present in an amount of 10 KE.

[0140] In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 20 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20 ±3, ±2, or ±1%, 25 ±3, ±2, or ±1%, 30 ±3, ±2, or ±1%, 35 ±3, ±2, or ±1%, 40 ±3, ±2, or ±1%, 45 ±3, ±2, or ±1%, or 50 ±3, ±2, or ±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 20 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 20 KE than when they are present in an amount of 10 KE.

[0141] In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 30 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20 ±3, ±2, or ±1%, 25 ±3, ±2, or ±1%, 30 ±3, ±2, or ±1%, 35 ±3, ±2, or ±1%, 40 ±3, ±2, or ±1%, 45 ±3, ±2, or ±1%, or 50 ±3, ±2, or ±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 30 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 30 KE than when they are present in an amount of 10 KE.

[0142] In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable S. pyogenes cells in the lyophilized composition is 20 ±3, ±2, or ±1%, 25 ±3, ±2, or ±1%, 30 ±3, ±2, or ±1%, 35 ±3, ±2, or ±1%, 40 ±3, ±2, or ±1%, 45 ±3, ±2, or ±1%, or 50 ±3, ±2, or ±1% higher when the nonviable S. pyogenes cells are present in the lyophilized composition in an amount of 40 KE than when they are present in an amount of 10 KE. In some embodiments, the relative percentage of nonviable Streptococcus pyogenes cells in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the nonviable Streptococcus pyogenes cells are present in the lyophilized composition in an amount of 40 KE than when they are present in an amount of 10 KE.

[0143] Immune checkpoint inhibitors The disclosed methods provide for the administration of a composition comprising nonviable S. pyogenes cells and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor may target the PD1 / PD-L1 / PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

[0144] In some embodiments, the immune checkpoint inhibitor is a small molecule, a nucleic acid molecule, a peptide, a protein, an antibody or its antigen-binding fragment, a fusion protein, a ribozyme, a vaccine, or a gene editing system.In some embodiments, the nucleic acid molecule is a gene therapy, a vaccine, or an inhibitory nucleic acid.In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide, an siRNA, an shRNA, or an miRNA.In some embodiments, the gene editing system is a CRISPR system, a TALEN system, or a ZFN system.

[0145] In certain embodiments, the immune checkpoint inhibitor targets the PD1 / PD-L1 / PD-L2 axis. In certain embodiments, the immune checkpoint inhibitor comprises a PD1 inhibitor and / or a PD-L1 inhibitor.

[0146] In certain embodiments, the composition comprising nonviable cells of Streptococcus pyogenes is administered in combination with a PD-1 inhibitor, e.g., a PD-1 specific antibody or binding fragment thereof, e.g., lambrolizumab, pidilizumab, nivolumab (Opdivo), or the like. TM , formerly known as MDX-1106), pembrolizumab (Keytruda TM , formerly known as MK-3475), cetrelimab (formerly known as JNJ 63723283), cemiplimab (Libtayo TM ), sintilimab (Tyvyt TM ), tislelizumab (Baizean TM ), toripalimab (Tuoyi TM ), penprimab (formerly known as AK105), dostallimab (Jemperli TM ), camrelizumab (Airuika TM , SHR-1210), prorugolimab (formerly BCD 100), pucotenlimab (HX008), selplulimab (HLX10), kadnilimab (Ketanil TM, anti-PD1 x anti-CTLA4 bispecific), gimverelimab (AB122), geptanolimab (GB226), nofazinlimab, sasanlimab (PF-06801591), QL-1604, phynotrimab (formerly SCT I10A), BAT-1306, budigalimab, ezabenlimab, peresolimab, pimivalimab, ruronilimab (formerly F520), spartalizumab, MK-3475A, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, IAP-0971, IBI-318 (anti-PD1 x anti-PD-L1 bispecific), ibonesimab (anti-PD-1 x anti-VEGFA bispecific antibody), tebotelimab (anti-PD-1 x anti-LAG3 bispecific antibody), AZD-2936 (anti-TIGIT x anti-PD-1 bispecific antibody), EMB-02 (anti-PD-1 x anti-LAG3 bispecific antibody), lorigerlimab (anti-PD-1 x anti-CTLA4 bispecific antibody), budalimab (anti-PD-1 x anti-CTLA4 bispecific antibody), volustomig (anti-PD-1 x anti-CTLA4 bispecific antibody), fidasimtamab (anti-PD-1 x anti-HER2 bispecific antibody), izlarimab (anti-PD-1 x anti-ICOS bispecific antibody), RG-6139 (anti-PD-1 x anti-LAG3 bispecific antibody), or any combination thereof.

[0147] In one embodiment, the composition comprising nonviable cells of Streptococcus pyogenes is administered with a PD-L1-specific antibody or binding fragment thereof, e.g., BMS-936559, durvalumab (Imfinzi), or a combination thereof. TM , MEDI4736), atezolizumab (Tecentriq TM , RG7446), avelumab (Bavencio TM , MSB0010718C), envafolimab (Enweida TM , KN035), sugemalimab (Cejemly TM), cosibelimab (CK-301), socazolimab (STI A1014), tagitanlimab (HBM 9167 or KL A167), MPDL3280A, SHR-1316, APL-502 (TQB 2450 or CBT 502), dumblestogg, betifisolimab, resabelimab, pakmilirumab, sudubrilimab (HS-636), LP-002, vintrafusp alfa (anti-PD-L1 antibody / TGFβRII extracellular domain fusion protein), SHR-1701 (anti-PD-L1 antibody / TGFβRII extracellular domain fusion protein), IBI-318 (anti-PD1 x anti-PD-L1 bispecific antibody), KN-046 (anti-PD-L1 x anti-CTLA4 bispecific antibody), 6MW-3211 (anti-CD47 x anti-PD-L1 bispecific antibody), BNT-311 (anti-PD-L1 x anti-4-1BB bispecific antibody), emfizatamab (anti-CD3e, anti-CD-19 anti-PD-L1, anti-4-1BB tetraspecific antibody), HB-0036 (anti-PD-L1 x anti-TIGIT bispecific antibody), HLX-301 (anti-TIGIT x anti-PD-L1 bispecific), or any combination thereof.

[0148] In one embodiment, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a PD-L1 inhibitor, e.g., a vaccine, such as IO102 / IO103 (IDO peptide + PD-L1 peptide vaccine) or mRNA-4359 (IDO peptide + PD-L1 mRNA vaccine).

[0149] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, tebotelimab (anti-PD-1 x anti-LAG3 bispecific antibody), RG-6139 (anti-PD-1 x anti-LAG3 bispecific antibody), or any combination thereof.

[0150] In some embodiments, the composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a CTLA4 inhibitor. In certain embodiments, the modified immune cells are used in combination with a CTLA4-specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, tuvonralimab, CTLA4-Ig fusion protein (e.g., abatacept, belatacept), kadonilimab (anti-PD1 x anti-CTLA4 bispecific antibody), KN-046 (anti-PD-L1 x anti-CTLA4 bispecific antibody), lorijelimab (anti-PD-1 x anti-CTLA4 bispecific antibody), budalimab (anti-PD-1 x anti-CTLA4 bispecific antibody), vollustomig (anti-PD-1 x anti-CTLA4 bispecific antibody), or any combination thereof.

[0151] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a B7-H3-specific antibody or binding fragment thereof, eg, enoblituzumab (MGA271), 376.96, or both.

[0152] In certain embodiments, compositions comprising nonviable cells of S. pyogenes are used in combination with a B7-H4-specific antibody or binding fragment thereof, such as an scFv or fusion protein thereof, as described, for example, in Dangaj et al., Cancer Res. 73:4820, 2013 and U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO 2016 / 40724 and WO 2013 / 025779, each of which is incorporated herein by reference in its entirety.

[0153] In some embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a CD244 inhibitor.

[0154] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a BLTA inhibitor, an HVEM inhibitor, a CD160 inhibitor, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO 2010 / 084158, the entire contents of which are incorporated herein by reference.

[0155] In a further embodiment, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a TIM3 inhibitor.

[0156] In yet a further embodiment, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a Gal9 inhibitor.

[0157] In some embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with an adenosine signaling inhibitor, such as a decoy adenosine receptor.

[0158] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with an A2aR inhibitor.

[0159] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a KIR inhibitor, such as lirilumab (BMS-986015).

[0160] In certain embodiments, a composition comprising nonviable cells of S. pyogenes is used in combination with an inhibitory cytokine (generally a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0161] In some embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with an IDO inhibitor, such as levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, IO102 / IO103 (IDO peptide + PD-L1 peptide vaccine), or any combination thereof.

[0162] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with an arginase inhibitor, such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0163] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a VISTA inhibitor, such as CA-170 (Curis, Lexington, Mass.).

[0164] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a LAIR1 inhibitor.

[0165] In certain embodiments, a composition comprising nonviable cells of Streptococcus pyogenes is used in combination with a CEACAM-1 inhibitor, a CEACAM-3 inhibitor, a CEACAM-5 inhibitor, or any combination thereof.

[0166] Treatment method Triple-negative breast cancer The present disclosure provides a method of treating triple-negative breast cancer in a subject, the method comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0167] In some embodiments, the subject is a human or non-human animal, such as a non-human primate, cow, horse, sheep, pig, cat, dog, goat, mouse, rat, rabbit, or guinea pig. In some embodiments, the subject is a human, such as a human adult, adolescent, child, or infant.

[0168] In some embodiments, the triple-negative breast cancer can be localized, regional, or metastatic. In some embodiments, the triple-negative breast cancer can be a newly diagnosed or recurrent cancer.

[0169] Triple-negative breast cancers can be classified into six subgroups: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR).

[0170] In some embodiments, the triple-negative breast cancer exhibits complete or partial resistance to a PD1 inhibitor or a PD-L1 inhibitor.

[0171] Biological samples can be collected from subjects to determine the presence and / or level of estrogen receptor, progesterone receptor, and HER2, or triple-negative state.As used herein, "biological sample" can be a biopsy specimen, a blood sample (serum or plasma can be prepared from the blood sample), body fluids (e.g., lung lavage fluid, ascites, mucosal lavage fluid, synovial fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation from a subject or biological source.Biological samples can also be collected from subjects before administering any composition containing non-viable cells of Streptococcus pyogenes.

[0172] Pharmaceutical compositions can be administered in a manner suitable for the disease or condition to be treated (or prevented), as determined by those skilled in the pharmaceutical field.The appropriate dosage and suitable administration period and frequency of the composition are determined by factors such as the patient's health condition, the patient's size (i.e., body weight, mass, or body surface area), the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration.In general, an appropriate dosage and treatment regimen provides the composition in an amount sufficient to bring about therapeutic and / or preventive benefits (e.g., those described herein, including improved clinical outcomes, such as increasing the frequency of complete or partial remission, or extending disease-free survival and / or overall survival, or reducing the severity of symptoms).For preventive use, the dosage is sufficient to prevent, delay the onset of, or reduce the severity of the disease or disorder associated with the disease. The prophylactic benefit of the immunogenic compositions administered in accordance with the methods described herein can be determined by conducting preclinical studies (including in vitro and in vivo animal studies) and clinical trials and analyzing the data obtained therefrom using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by one of ordinary skill in the art.

[0173] The pharmaceutical compositions described herein can be in unit dose or multi-dose containers, for example, sealed ampoules or vials.Such containers can be frozen to maintain the stability of formulations until use.The specific compositions described herein can be used in various treatment regimens, including, for example, parenteral administration or intravenous administration or formulation, and develop suitable administration regimens and treatment regimens.

[0174] The composition comprising nonviable S. pyogenes can be administered based on milligrams of dried cell mass, or KE. Thus, it can be expressed in either mg or KE. In certain embodiments, the dose of the composition comprising nonviable S. pyogenes is about 0.1 KE to about 200 KE, about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1 KE, about 0.5 KE, about 1 KE, about 2.5 KE, about 5 KE, about 10 KE, about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, about 60 KE, about 70 KE, about 80 KE, about 90 KE, about 100 KE, about 125 KE, about 150 KE, about 175 KE, or about 200 KE. In certain embodiments, the unit dose comprising the composition comprising nonviable Streptococcus pyogenes is about 0.01 mg to about 20 mg, or about 0.01 mg, about 0.025 mg, about 0.05 mg, about 0.075 mg, about 0.1 mg, about 0.125 mg, about 0.150 mg, about 0.175 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg.

[0175] In some embodiments, the composition comprising nonviable Streptococcus pyogenes is administered to the subject once daily, twice weekly, once weekly, biweekly, or once monthly.

[0176] When the subject compositions are administered parenterally, they may also comprise sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in a mixture with water. The aqueous solution or suspension may further contain one or more buffers, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used in preparing any unit-dose formulation is pharmaceutically pure and substantially non-toxic in the amounts used. Furthermore, the active compound may be incorporated into sustained-release preparations and formulations. As used herein, unit-dose form refers to a physically discrete unit suitable as a single dose for the subject to be treated; each unit may contain a predetermined amount of recombinant cells or active compound calculated to produce the desired therapeutic effect, together with an appropriate pharmaceutical carrier.

[0177] Generally, appropriate dosage and treatment regimen provides active molecules or cells in sufficient amount to bring about therapeutic or preventive benefits.Such response can be monitored by establishing the improved clinical outcome (for example, the increased frequency of complete or partial remission, or the prolonged disease-free survival period) in treated subjects compared with untreated subjects.The increase in the existing immune response to tumor protein generally correlates with the improved clinical outcome.Such immune response can generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which can be carried out using samples obtained from subjects before and after treatment.

[0178] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted prior to administration, e.g., to form a liquid formulation of the present disclosure.

[0179] In some embodiments, the formulation is administered to the subject using conventional delivery modes, including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intrathecal, intramuscular, subcutaneous, or intratumoral administration.

[0180] In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject prior to the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days or more before the immune checkpoint inhibitor. In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject concurrently with the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered on the same day as the immune checkpoint inhibitor. In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject subsequent to the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days or more after the immune checkpoint inhibitor.

[0181] In other embodiments, the methods of the present disclosure further comprise administering an additional treatment comprising one or more of: an antibody or antigen-binding fragment specific for a cancer antigen expressed by the targeted solid tumor; a small molecule, a chemotherapeutic agent; surgery; radiation therapy treatment; a cytokine; RNA interference therapy, or any combination thereof.

[0182] Exemplary monoclonal antibodies useful in cancer therapy include, for example, those described in Galluzzi et al., Oncotarget 5(24):12472-12508, 2014, which are incorporated by reference in their entirety.

[0183] In some embodiments, the combination therapy further comprises subjecting the patient to radiation therapy or surgery.Radiation therapy includes X-ray therapy such as gamma irradiation and radiopharmaceutical therapy.Surgery and surgical techniques suitable for treating certain cancers or non-inflammatory solid tumors can be used in subjects in combination with the modified immune cells of the present disclosure.

[0184] In some embodiments, the combination therapy further comprises administering to the subject chemotherapeutic agent.The chemotherapeutic agent includes but is not limited to chromatin function inhibitor, topoisomerase inhibitor, microtubule inhibitor, DNA damage agent, metabolic antimetabolite (for example, folic acid antagonist, pyrimidine analog, purine analog, and sugar modification analog), DNA synthesis inhibitor, DNA interaction agent (for example, intercalating agent), and DNA repair inhibitor. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, Epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolomide, teniposide, triethylenethiophosphoramide, and etoposide (VP Antiproliferative / mitotic inhibitors, including 16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes it from cells that do not have the ability to synthesize it themselves); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes - dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and others) thrombin inhibitors); fibrinolytics (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP47 0, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin) cin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prednisolone);Growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0185] Cytokines can be used to manipulate host immune responses for anti-cancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42:539, 2015. Cytokines useful for promoting anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, either alone or in any combination.

[0186] Another cancer treatment approach involves reducing the expression of oncogenes and other genes necessary for the growth, maintenance, proliferation, and immune evasion of cancer cells. RNA interference, particularly the use of microRNAs (miRNAs) and small inhibitory RNAs (siRNAs), provides an approach to knock down the expression of oncogenes. See, for example, Larsson et al., Cancer Treat. Rev. 16:128, 2017.

[0187] In any of the embodiments disclosed herein, any of the therapeutic agents may be administered to a subject one or more times over the course of treatment, and when combined, they may be administered to a subject in any order (e.g., simultaneously, in parallel, or in any order) or in any combination. The appropriate dose, appropriate duration, and frequency of administration of the composition will be determined by factors such as the patient's condition; the size, type, spread, growth, and severity of the tumor or cancer; the particular form of the active ingredient; and the method of administration.

[0188] An effective amount of a therapeutic or pharmaceutical composition refers to a sufficient amount, at the dosage and for the period of time necessary, to achieve the desired clinical result or beneficial treatment, as described herein.An effective amount can be delivered in one or more administrations.When the administration is to a subject who is already known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used in relation to treatment, while a "prophylactically effective amount" can be used to describe administering an effective amount to a subject who is susceptible to or at risk of developing a disease or disease state (e.g., recurrence) as a preventative measure.

[0189] Non-muscle-invasive bladder cancer The present disclosure provides a method of treating non-muscle-invasive bladder cancer in a subject, the method comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0190] In some embodiments, the subject is a human or non-human animal, such as a non-human primate, cow, horse, sheep, pig, cat, dog, goat, mouse, rat, rabbit, or guinea pig. In some embodiments, the subject is a human, such as a human adult, adolescent, child, or infant.

[0191] Bladder cancer can be classified according to the traditional American Joint Committee on Cancer (AJCC) TNM staging system. In the absence of lymph node metastasis (N stage) or distant metastasis (M stage), the depth of tumor invasion (T stage) is the most important determinant, and tumors can be dichotomized based on whether they invade the muscularis propria (muscle-invasive bladder cancer, MIBC) or not (non-muscle-invasive bladder cancer, NMIBC). Table B shows the AJCC primary tumor staging (T) for bladder cancer. Tumors can be further classified by histologic grade (low or high). The World Health Organization (WHO) / International Society of Urological Pathology (ISUP) 2004 classification of non-muscle-invasive urothelial neoplasms is shown in Table C. The World Health Organization (WHO) 2004 grading system for urothelial carcinoma is shown in Table D. [Table 2] [Table 3] [Table 4]

[0192] In some embodiments, the subject with non-muscle invasive bladder cancer has Ta stage tumor. In some embodiments, the subject with non-muscle invasive bladder cancer has T1 stage tumor. In some embodiments, the subject with non-muscle invasive bladder cancer has Tis stage (CIS) tumor. In some embodiments, CIS tumor may or may not be accompanied by Ta and / or T1.

[0193] In some embodiments, the subject has low-grade papillary urothelial neoplasm (PUNLMP). In some embodiments, the subject has low-grade non-muscle-invasive bladder cancer. In some embodiments, the subject has high-grade non-muscle-invasive bladder cancer. In some embodiments, the subject has high-grade Ta non-muscle-invasive bladder cancer. In some embodiments, the subject has high-grade T1 non-muscle-invasive bladder cancer.

[0194] Non-muscle-invasive bladder cancer can also be classified into three different risk categories based on the American Urological Association (AUA) and / or European Association of Urology (EAU) guidelines. The risk stratification groups and criteria for NMIBC are shown in Table E. [Table 5] LG = low grade; PUNLMP = low-grade papillary urothelial neoplasm; HG = high grade; CIS = carcinoma in situ; LVI = lymphovascular invasion

[0195] In some embodiments, the subject has non-muscle invasive bladder cancer that is identified as low risk according to AUA and / or EUA guideline.In some embodiments, the subject has non-muscle invasive bladder cancer that is identified as moderate risk according to AUA and / or EUA guideline.In some embodiments, the subject has non-muscle invasive bladder cancer that is identified as high risk according to AUA and / or EUA guideline.In some embodiments, the subject has non-muscle invasive bladder cancer that is identified as highest risk according to EUA guideline.In some embodiments, the subject with non-muscle invasive bladder cancer has lymphatic invasion.

[0196] Intravesical Bacillus Calmette-Guerin (BCG) is a standard therapy for treating high-risk non-muscle-invasive bladder cancer in BCG-naive patients, such as after surgical resection or ablation of the tumor. In some embodiments, the subject with non-muscle-invasive bladder cancer has not previously received BCG therapy (BCG-naive). In some embodiments, the subject with non-muscle-invasive bladder cancer has received appropriate BCG treatment. In some embodiments, the subject with non-muscle-invasive bladder cancer is refractory to BCG therapy. Definitions of BCG-refractory disease and appropriate BCG treatment are provided in Table F. [Table 6]

[0197] Other terms describing the clinical scenario in which BCG is not effective in treating high-risk non-muscle invasive bladder cancer and is no longer a treatment option include BCG failure (when muscle invasive bladder cancer is detected), BCG-refractory (when high-risk lesions are detected during or after adequate treatment at 3 or 6 months of treatment), BCG-relapsing (when tumors are detected after the completion of treatment, after the initial response), and BCG inadequately treated (when patients do not receive full BCG treatment due to intolerance to BCG drugs or unavailability of BCG drugs).In some embodiments, the subject with non-muscle invasive bladder cancer has BCG-failure NMIBC.In some embodiments, the subject with non-muscle invasive bladder cancer has BCG-refractory NMIBC.In some embodiments, the subject with non-muscle invasive bladder cancer has BCG-relapsing NMIBC. In certain embodiments, the subject with non-muscle-invasive bladder cancer was inappropriately treated with BCG.

[0198] In some embodiments, the subject with non-muscle-invasive bladder cancer has not undergone a radical cystectomy. In some embodiments, the subject with non-muscle-invasive bladder cancer is ineligible for radical cystectomy.

[0199] In some embodiments, the non-muscle-invasive bladder cancer can be a newly diagnosed cancer or a recurrent cancer.

[0200] In some embodiments, the non-muscle-invasive bladder cancer exhibits complete or partial resistance to PD1 or PD-L1 inhibitors.

[0201] To determine the presence and / or stage or risk level of non-muscle invasive bladder cancer, a biological sample can be collected from the subject. As used herein, a "biological sample" can be a biopsy specimen, a blood sample (from which serum or plasma can be prepared), a body fluid (e.g., urine, mucosal washings), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation from a subject or biological source. A biological sample can also be collected from a subject before administering any composition containing non-viable cells of Streptococcus pyogenes.

[0202] Pharmaceutical compositions can be administered in a manner suitable for the disease or condition to be treated (or prevented), as determined by those skilled in the pharmaceutical field.The appropriate dosage and suitable administration period and frequency of the composition are determined by factors such as the patient's health condition, the patient's size (i.e., body weight, mass, or body surface area), the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration.In general, an appropriate dosage and treatment regimen provides the composition in an amount sufficient to bring about therapeutic and / or preventive benefits (e.g., those described herein, including improved clinical outcomes, such as increasing the frequency of complete or partial remission, or extending disease-free survival and / or overall survival, or reducing the severity of symptoms).For preventive use, the dosage is sufficient to prevent, delay the onset of, or reduce the severity of the disease or disorder associated with the disease. The prophylactic benefit of the immunogenic compositions administered in accordance with the methods described herein can be determined by conducting preclinical studies (including in vitro and in vivo animal studies) and clinical trials and analyzing the data obtained therefrom using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by one of ordinary skill in the art.

[0203] The pharmaceutical compositions described herein can be in unit dose or multi-dose containers, for example, sealed ampoules or vials.Such containers can be frozen to maintain the stability of formulations until use.The specific compositions described herein can be used in various treatment regimens, including, for example, parenteral administration or intravenous administration or formulation, and develop suitable administration regimens and treatment regimens.

[0204] The composition comprising nonviable S. pyogenes can be administered based on milligrams of dried cell mass, or KE. Thus, it can be expressed in either mg or KE. In certain embodiments, the dose of the composition comprising nonviable S. pyogenes is about 0.1 KE to about 200 KE, about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1 KE, about 0.5 KE, about 1 KE, about 2.5 KE, about 5 KE, about 10 KE, about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, about 60 KE, about 70 KE, about 80 KE, about 90 KE, about 100 KE, about 125 KE, about 150 KE, about 175 KE, or about 200 KE. In certain embodiments, the unit dose comprising the composition comprising nonviable Streptococcus pyogenes is about 0.01 mg to about 20 mg, or about 0.01 mg, about 0.025 mg, about 0.05 mg, about 0.075 mg, about 0.1 mg, about 0.125 mg, about 0.150 mg, about 0.175 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg.

[0205] In some embodiments, the composition comprising nonviable Streptococcus pyogenes is administered to the subject once daily, twice weekly, once weekly, biweekly, or once monthly.

[0206] When the subject compositions are administered parenterally, they may also comprise sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in a mixture with water. The aqueous solution or suspension may further contain one or more buffers, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used in preparing any unit-dose formulation is pharmaceutically pure and substantially non-toxic in the amounts used. Furthermore, the active compound may be incorporated into sustained-release preparations and formulations. As used herein, unit-dose form refers to a physically discrete unit suitable as a single dose for the subject to be treated; each unit may contain a predetermined amount of recombinant cells or active compound calculated to produce the desired therapeutic effect, together with an appropriate pharmaceutical carrier.

[0207] Generally, appropriate dosage and treatment regimen provides active molecules or cells in sufficient amount to bring about therapeutic or preventive benefits.Such response can be monitored by establishing the improved clinical outcome (for example, the increased frequency of complete or partial remission, or the prolonged disease-free survival period) in treated subjects compared with untreated subjects.The increase in the existing immune response to tumor protein generally correlates with the improved clinical outcome.Such immune response can generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which can be carried out using samples obtained from subjects before and after treatment.

[0208] In some embodiments, the lyophilized pharmaceutical formulation is reconstituted prior to administration, e.g., to form a liquid formulation of the present disclosure.

[0209] In some embodiments, the formulation is administered to the subject using conventional delivery modes, including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intrathecal, intramuscular, subcutaneous, or intratumoral administration.

[0210] In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject prior to the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days or more before the immune checkpoint inhibitor. In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject concurrently with the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered on the same day as the immune checkpoint inhibitor. In some embodiments, the composition comprising nonviable Streptococcus pyogenes cells is administered to a subject subsequent to the immune checkpoint inhibitor. For example, the composition comprising nonviable Streptococcus pyogenes cells can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days or more after the immune checkpoint inhibitor.

[0211] In other embodiments, the methods of the present disclosure further comprise administering an additional treatment comprising one or more of: an antibody or antigen-binding fragment specific for a cancer antigen expressed by the targeted solid tumor; a small molecule, a chemotherapeutic agent; surgery; radiation therapy treatment; a cytokine; RNA interference therapy; a cancer vaccine, or any combination thereof.

[0212] Exemplary monoclonal antibodies useful in cancer therapy include, for example, those described in Galluzzi et al., Oncotarget 5(24):12472-12508, 2014, which are incorporated by reference in their entirety.

[0213] In some embodiments, the combination therapy further comprises subjecting the patient to radiation therapy or surgery.Radiation therapy includes X-ray therapy such as gamma irradiation and radiopharmaceutical therapy.Surgery and surgical techniques suitable for treating certain cancers or non-inflammatory solid tumors can be used in subjects in combination with the modified immune cells of the present disclosure.

[0214] In some embodiments, the combination therapy further comprises administering BCG therapy to the subject.

[0215] In some embodiments, the combination therapy further comprises administering to the subject chemotherapeutic agent.The chemotherapeutic agent includes but is not limited to chromatin function inhibitor, topoisomerase inhibitor, microtubule inhibitor, DNA damage agent, metabolic antimetabolite (for example, folic acid antagonist, pyrimidine analog, purine analog, and sugar modification analog), DNA synthesis inhibitor, DNA interaction agent (for example, intercalating agent), and DNA repair inhibitor. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, Epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolomide, teniposide, triethylenethiophosphoramide, and etoposide (VP Antiproliferative / mitotic inhibitors, including 16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes it from cells that do not have the ability to synthesize it themselves); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes - dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and others) thrombin inhibitors); fibrinolytics (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP47 0, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin) cin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prednisolone);Growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0216] Cytokines can be used to manipulate host immune responses for anti-cancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42:539, 2015. Cytokines useful for promoting anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, either alone or in any combination.

[0217] Another cancer treatment approach involves reducing the expression of oncogenes and other genes necessary for the growth, maintenance, proliferation, and immune evasion of cancer cells. RNA interference, particularly the use of microRNAs (miRNAs) and small inhibitory RNAs (siRNAs), provides an approach to knock down the expression of oncogenes. See, for example, Larsson et al., Cancer Treat. Rev. 16:128, 2017.

[0218] In any of the embodiments disclosed herein, any of the therapeutic agents may be administered to a subject one or more times over the course of treatment, and when combined, they may be administered to a subject in any order (e.g., simultaneously, in parallel, or in any order) or in any combination. The appropriate dose, appropriate duration, and frequency of administration of the composition will be determined by factors such as the patient's condition; the size, type, spread, growth, and severity of the tumor or cancer; the particular form of the active ingredient; and the method of administration.

[0219] An effective amount of a therapeutic or pharmaceutical composition refers to a sufficient amount, at the dosage and for the period of time necessary, to achieve the desired clinical result or beneficial treatment, as described herein.An effective amount can be delivered in one or more administrations.When the administration is to a subject who is already known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used in relation to treatment, while a "prophylactically effective amount" can be used to describe administering an effective amount to a subject who is susceptible to or at risk of developing a disease or disease state (e.g., recurrence) as a preventative measure. [Example]

[0220] Example 1: In vivo efficacy of Streptococcus pyogenes nonviable cells in a triple-negative EMT6 breast cancer model as monotherapy and in combination with anti-mPD-1

[0221] material and method Animals. Sixty-five 7-week-old female Balb / c mice were purchased from Jackson Laboratories and housed five mice per cage in our in-house animal facility upon arrival, with free access to food and water. Fifty-four mice were enrolled in the study at the time of tumor randomization; the remaining 11 mice (which received tumor cell injections) were used solely for randomization purposes. After a one-week acclimation period, all mice found to be healthy were weighed on day 0, prior to tumor cell implantation, and twice weekly thereafter until the end of the study.

[0222] Treatment. Composition 002 ("Comp. 002" or "002") was received as lyophilized material in individual vials in a refrigerated box several days prior to the start of the study. There were 49 vials, each containing 20 KE, corresponding to a drug dose of 2 mg. Composition 002 is a lyophilized biological preparation for administration containing nonviable cells of Streptococcus pyogenes (Group A, Type 3) Su strain treated with hydrogen peroxide and benzylpenicillin in accordance with the present disclosure. Composition 002 is manufactured using the same master cell bank as OK-432 (Picibanil®).

[0223] All vials were stored in a refrigerator (4°C) at the recommended temperature of 2-8°C. Before administration to mice on each treatment day, each vial was freshly reconstituted with 1 ml of 0.9% sterile saline. Any remaining solution in the dosing vial was discarded after each administration.

[0224] Anti-PD1 antibody (CD279, clone RMP1-14, cat# BP0146) and the corresponding IgG control (isotype rat IgG2a,k (clone 2A3, cat# BP0089)) were purchased from BioXcell. Due to a shipping delay of several days, mice were administered several days after the scheduled administration time.

[0225] Tumor cells and in vitro culture. EMT6 mouse triple-negative breast cancer cells were thawed from a frozen vial and cultured in sterile DMEM medium supplemented with sterile 10% FBS and incubated at 37°C in a humidified atmosphere of 5% CO. Cells were passaged at a constant split ratio of approximately 1:6 during culture, meaning that cells were consistently harvested and replated into new T75 culture flasks at the same confluence (approximately 70-80%) and in exponential growth phase at each passage.

[0226] Cell culture and harvest conditions were maintained under standardized conditions to minimize any variability. Cells were passaged two days before the scheduled injection into mice. On each occasion and on the day of transplantation, cells were harvested by brief treatment (1-2 minutes) with warmed trypsin / EDTA solution, followed by the addition of sterile 10% serum-containing culture medium and two washes with sterile serum-free culture medium.

[0227] The cell suspension was kept on ice during preparation, up to the time of injection, and between injections to maintain cell viability and adherence. After the final centrifugation, cells were counted and 2x10 5 For injection of cells / mouse, 2.0x10 cells / mouse in sterile serum-free culture medium 6 The cells were appropriately resuspended to a final concentration of 1 / ml. The calculated cell viability was 99%, confirming that highly viable cells were transplanted into the mice.

[0228] Sample processing for flow cytometry (FACS) analysis of SPLC and TIL: Spleens were collected from a subset of three mice per group under sterile conditions and placed in cold sterile medium. A single-cell suspension of splenocytes (SPLC) was prepared by pressing the spleen with the plunger of a 3 ml syringe and passing it through a 40 μm cell strainer. SPLC were then processed and stained for FACS analysis.

[0229] Tumors were also collected from the same subset of mice (3 mice per group), and tumor-infiltrating lymphocytes (TILs) were prepared by mechanical dissociation and then treated with freshly prepared collagenase D enzyme at a final concentration of 2.5 mg / ml for 30 minutes immediately before use. The cell suspension was filtered through a 70 μm cell strainer, washed with sterile HBSS containing 2% FBS, and stained for FACS analysis.

[0230] Staining antibodies and flow cytometry analysis. Antibody panels and other related reagents for flow cytometry were pre-purchased from BioLegend, BD Bioscience, and ThermoFisher Scientifics before sample processing and staining for FACS analysis.

[0231] Immunophenotyping (i.e., CD8 + T cells, regulatory CD4 + The selected staining panel for T cells, NK cells, myeloid-derived suppressor cells (MDSC), Tregs and macrophages) and FACS analysis is described in the table below.

[0232] All samples were taken from the LSRFortessa TM (BD Biosciences) flow cytometer. FACS profiles were further analyzed using FloJo software (treeStar). [Table 7]

[0233] Single cell preparation for scRNA-seq: At the end of the study, three selected tumors were processed for scRNA-seq: one tumor from the intravenous control group (Group 2, Mouse #4), a second tumor from the Composition 002, 10 mg / Kg intravenous group (Group 3, Mouse #6), and a third tumor from the Composition 002, 10 mg / Kg intratumoral group (Group 4, Mouse #6).

[0234] Tumors were mechanically and enzymatically dissociated into single cells using collagenase type IV (final concentration 1 mg / ml) and DNAse (final concentration 100 units / ml) in an incubator at 37°C and 5% CO2 for approximately 1 hour. The samples were then gently washed with HBSS containing 2% FBS by centrifugation at 980 rpm, treated with ACK on ice for 3 minutes, washed again, processed through a cell death removal kit, and then resuspended in DMEM containing 20% ​​FBS and 10% DMSO for cryopreservation. 2.5 x 10 6 cells (vial #1) and 5.0 x10 6 There were two vials (1 ml each) of each sample containing cells (vial #2). All samples contained single, highly viable cells (average >98%).

[0235] Samples were stored frozen until scRNA-seq analysis.

[0236] Study design and results In vivo implantation of tumor cells and treatment of tumor-bearing mice. After a one-week acclimation period, 2 x 10 cells in a volume of 100 μl were injected into the left fourth mammary fat pad of all 65 anesthetized mice. 5 The number of cells injected in this study was 5 x 10, the number used in previous studies. 5 The dose was reduced from 100 mm to 120 mm, the purpose of which was to attenuate the rapid formation and growth of tumors and prevent early ulceration. This change was made when tumors were approximately 100 mm in size before treatment began. 3This was valuable because it allowed mice to be randomized at different times, allowing for more accurate and less variable efficacy results.

[0237] Mice were closely monitored twice weekly, and tumor growth was also assessed twice weekly by measuring length (L) and width (W) with a vernier caliper. Tumor size was calculated by the formula (L x W2) / 2). Almost all tumors were measurable with a vernier caliper 6 days after implantation, and their average size reached approximately 100 mm by 8 days. 3 The tumor take rate at this time point was 97%. Pre-randomization tumor growth data are expressed as mean + SEM and are shown graphically in Figure 1.

[0238] Eight days after tumor cell injection, 54 mice bearing tumors of comparable size were randomized into nine groups of six mice per group to ensure that tumor means and standard deviations were similar between groups prior to treatment exposure. The remaining 11 mice with tumors too small (or too large to measure with calipers) or too large were not enrolled. After randomization, mice in Groups 1 and 2 (control groups) were administered saline vehicle intratumorally and intravenously, respectively. Mice in Group 6 were administered anti-PD-1 antibody (200 μg / mouse, 100 μl volume) intraperitoneally (ip) as a single agent, and mice in the control groups, Groups 1 and 2, were administered an IgG control at the same dose and volume intraperitoneally. Mice in Groups 4 and 8 were administered Composition 002 intravenously at a dose of 10 mg / Kg (100 μl volume), alone and in combination with the anti-PD-1 antibody, respectively.

[0239] Mice in treatment groups 3, 5, 7, and 9 were administered Composition 002 at doses of 10 mg / Kg and 20 mg / Kg by the intratumoral (it) route as a single agent (Groups 3 and 5) and in combination with an anti-PD-1 antibody (Groups 7 and 9).

[0240] Mice in groups 5 and 9 were initially planned to receive Composition 002 intravenously (iv) at a dose of 20 mg / kg. However, this high intravenous dose was not tolerated, resulting in the death of one mouse that exhibited convulsions immediately after treatment. The dose was subsequently reduced to 15 mg / kg, but still elicited several adverse reactions: some mice exhibited decreased activity, ruffled fur, and weight loss, which resolved within 2–3 days, while several other mice experienced more severe symptoms. Tests with several additional mice confirmed that 20 mg / kg of Composition 002 was tolerated when administered intratumorally, and it was decided to deliver this dose exclusively intratumorally. Therefore, mice in groups 5 and 9 initially received a single intravenous dose of 15 mg / kg, followed by continued administration at 20 mg / kg via the intratumoral route.

[0241] The Composition 002 treatment schedule for all mice was twice weekly, on Mondays and Thursdays, for three weeks. The schedule for anti-PD-1 administration was also twice weekly, one day after treatment with Composition 002. The treatment groups are listed in Table 1 below. [Table 8]

[0242] The volume of drug delivered into the tumor was 50 μl during the first week and then increased to 100 μl as tumor size increased. The 100 μl volume was maintained constant in all other groups throughout the study. To improve intratumoral delivery by providing better penetration and uniform distribution of the drug, a multi-sided needle (purchased from Cook Medical) was planned. However, when the needle was used with saline in two additional mice, its size and thickness made it difficult to insert without damaging the skin, and its performance was very poor, even though it was the smallest available.

[0243] After treatment initiation, tumor growth continued to be assessed twice weekly by caliper measurement. Data are expressed as tumor volume mean + standard deviation (STDV) and standard error (SEM), and growth curves (mean + SEM) for all groups are shown graphically in Figure 2.

[0244] The percent tumor growth inhibition (%TGI) in the treatment groups was calculated and the results are shown in Table 2. [Table 9]

[0245] Tumor efficacy data obtained from intravenous and intratumoral administration of Composition 002 are also shown in separate graphical representations as mean + SEM in Figures 3A and 3B, respectively.

[0246] Similar to the results of our previous efficacy study (unpublished), the data from this study indicate that Composition 002 at a dose of 10 mg / kg inhibited EMT6 tumor growth more effectively when delivered intravenously than when delivered intratumorally. Furthermore, in combination with an anti-PD1 antibody, systemic delivery of Composition 002 at 10 mg / kg demonstrated superior antitumor activity to intratumoral administration of either 10 or 20 mg / kg. Furthermore, intratumoral treatment with Composition 002 did not induce a dose-response pattern, recapitulating previous observations. Due to the adverse events described above, a higher dose of 20 mg / kg could not be evaluated for the intravenous delivery route.

[0247] Thirty days after tumor cell implantation, i.e., 21 days after the start of treatment, primary tumors and spleens were collected from three mice in each group, processed, and stained for FACS analysis. Interestingly, mouse #2 in Group 8 (Composition 002 intravenous + anti-PD-1) had a tumor mass of 130 mm on the 15th day after the start of treatment. 3 The tumors measured 100x the normal size and gradually decreased in size until they were no longer palpable by the end of the study. Spleens were collected from these mice and analyzed by FACS.

[0248] A total of three tumors were collected and dissociated into single cells for scRNA analysis. Interestingly, mouse #2 in group 8 (Composition 002 intravenous + anti-PD-1) regressed to the point where it could not be measured with a caliper at the end of the study. For this mouse, only the spleen was collected and analyzed by FACS. Primary tumors and livers from the remaining mice were fixed in 10% neutral buffered formalin. Lungs were collected from all mice in all groups and fixed in formalin. Details of the euthanasia date and processing of collected samples are summarized in Table 3. [Table 10-1] [Table 10-2] [Table 10-3]

[0249] Except for one metastasis measuring 1-2 mm on the lung surface of mouse #2 in Group 4 (Composition 002 10 mg / Kg, IV), no metastases were visible to the naked eye in the lungs of mice in all groups, but histological confirmation is required.

[0250] Fixed samples were processed for paraffin embedding, and blocks from all samples were prepared by the histopathology laboratory.

[0251] To comply with IACUC regulations, several mice were euthanized several days before the end of the study due to large tumor growth and the development of dark eschars, an indication of the onset of ulceration. As shown by the data, compared to our previous (unpublished) study, fewer tumor cells were implanted this time, resulting in less aggressive tumor growth and a reduced number of large, ulcerated tumors. Both tumors and spleens were weighed, and the weight data are presented as mean + SEM and are graphically displayed in Figures 4A-4B (intratumoral delivery group) and Figures 5A-5B (intravenous delivery group). Interestingly, an inverse correlation in tumor and spleen size was found: mice that responded better to Composition 002 had reduced tumor growth and larger spleens. This result was more pronounced and statistically significant in the IV delivery group, which showed the best response to 10 mg / kg Composition 002 in combination with anti-PD-1, and which had the most enlarged spleens, indicating a stronger systemic immune response.

[0252] Monitoring Mouse Weight and Health. Throughout the study, mice were closely monitored daily for any abnormal signs, including respiratory distress, weakness, lethargy, lameness and / or weight changes (e.g., loss), large tumors (i.e., size limit >2,000), ulcerated tumors, metastatic burden, and death.

[0253] Body weight and health status information was recorded and provided in an Excel file every week.Body weight data was expressed as mean and percent change;Graphical representations of these data for all groups are shown in Figures 6A and 6B and Figures 7A and 7B, respectively, as pre-randomization and post-randomization.Separate graphical representations of the data for intratumoral and intravenous drug delivery groups are shown in Figures 7A and 7B, respectively.

[0254] None of the mice showed significant weight changes, i.e., weight loss of >15% or >20%, although accurate measurements were not possible in mice with the largest tumors or enlarged spleens because the increased tumor or spleen weight partially offset the weight loss. Two deaths occurred: one in mouse #2 in the control group of Group 2, and the other in mouse #6 in Group 8 (combination of Composition 002 10 mg / kg IV and anti-PD-1 antibody); the control mouse's death was likely due to a large tumor with dark crusts / ulcerations, while the other mouse's death was likely due to a combination of treatment effects, a strong immune response, or both.

[0255] 2,000 mm 3 Mice with tumors that reached or exceeded the size limit were euthanized within 24-48 h to allow for planned collection and analysis and to adhere to IACUC protocol requirements.

[0256] Flow cytometry analysis of splenocytes (SPLN) and tumor-infiltrating lymphocytes (TIL). After tumor (for TIL) and spleen (for SPLC) collection and processing, samples were stained and analyzed by flow cytometry to determine the effect of Composition 002 on various immune cell types according to the immunophenotyping and selected panels described above in the Methods section. Separate duplicate data and mean values ​​+ standard deviations for each sample are shown in Tables 4A-7C. The percentages of T cell subsets in the spleen and tumor-derived TIL were analyzed using a flow cytometer. Flow cytometry data for the spleen from mouse #2 in combination group 8 (Composition 002 10 mg / kg intravenously + anti-PD-1), which showed tumor regression, were similar to the mean values ​​for this group for all immunophenotypes. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22]

[0257] Graphical representations of the immune cell percentage data displayed as scatter plots are shown in Figures 9-19. These results compare two delivery routes of Composition 002 at a 10 mg / Kg dose. Statistical analysis to determine significant differences in expression of selected markers between groups was performed using unpaired t-tests and one-way ANOVA using GraphPad Prism 9.

[0258] The results shown in Figures 9A-9B are similar to data from our previous study, which showed minimal or statistically insignificant changes in the percentage of CD3+ T cells in all groups, both in splenocytes and tumor TILs from mice administered Composition 002 intravenously, and in tumors from mice administered the drug intratumorally. However, this time there was a consistent and significant increase in T cells in the spleens of mice administered Composition 002 intratumorally. Interestingly, however, this increase returned to baseline levels when Composition 002 was combined with an anti-PD-1 antibody. Composition 002 delivered intratumorally at a dose as high as 20 mg / Kg had no such effect.

[0259] The results shown in Figures 10A-10B, which demonstrate the effect of Composition 002 on the spleen and tumor, are relatively consistent with our previous observations: effects were observed in the spleen when the drug was delivered as a single agent or in combination with anti-PD-1 by either route, but there was only a trend toward an approximately 40-50% reduction in CD4+ T cells induced in the tumor by intravenous administration of the drug. In this study, the data demonstrated less variable, better, and statistically significant CD4+ T cell inhibition in the tumor after intravenous administration of Composition 002, which was sufficient to offset the increase caused by anti-PD-1 in the combination treatment.

[0260] As shown in Figures 11A-11B, the percentage of CD8+ T cells [having a CD45+CD3+CD4-CD8+ phenotype] was reduced in the spleen and tumor by intravenous administration of Composition 002, and the reduction in the spleen was statistically significant compared to both the control group and anti-PD-1 treatment. In the combination group in which Composition 002 was delivered intravenously, the increasing effect on CD8+ T cells induced by anti-PD-1 treatment alone was significantly attenuated by Composition 002, resulting in reduced levels of immune cells with this phenotype; this low level was comparable to that measured after treatment with Composition 002 alone. In tumors from mice treated intratumorally with Composition 002, Composition 002 alone did not have such a significant inhibitory effect, but there was a statistically significant synergistic effect of the combination of Composition 002 and anti-PD-1 compared to each single agent and the control group.

[0261] When examining the percentage of NK cells (gated as CD45+CD3-CD49b+CD335+) in splenocytes or TILs in all intravenous treatment groups, no significant changes were observed, except for a significant decrease in the spleen with single-agent treatment with Composition 002 or anti-PD-1 compared to the control group, which was further decreased in the combination group as shown in Figures 12A-12B. In the tumor, there was no difference between intravenous treatment with Composition 002 or single-agent treatment with PD-1 and the control group, but there was a statistically significant increase in NK cells in the combination group.

[0262] There was no difference in the percentage of NK cells in the spleen between the intratumoral groups, but there was a statistically significant reduction in the tumor with anti-PD1 in both the single agent and combination groups.

[0263] To assess the effect of treatment on the immunosuppressive microenvironment of EMT6 tumors, the percentages of granulocytic and monocytic MDSCs and the ratio of granulocytic to monocytic MDSCs were calculated from the analysis of the myeloid-derived suppressor cell subpopulations granulocytes and monocytes, and the data are shown in Figures 13A-13B and 14A-14B, respectively. These results show that in the tumor, this ratio was unaffected by Composition 002 but increased by anti-PD-1 antibodies, correlating with the lack of efficacy of the PD-1 inhibitor; however, in the combination group, this ratio was dramatically reduced, likely due to the indirect effect of Composition 002 delivered either intravenously or intratumorally. In contrast, these effects were not evident in the spleen, where Composition 002 increased the MDSC / monocyte ratio when administered alone to mice by either route, but not when administered in combination with anti-PD-1.

[0264] The phenotype CD45+CD3-CD4 is graphically depicted in Figures 15A-15B. + CD25 + Foxp3 + Data analysis of regulatory T cells (Tregs) with Composition 002 showed no significant change in the spleen with either intravenous or intratumoral administration, but a statistically significant increase in tumors after intravenous delivery, and a decrease with anti-PD-1 treatment. The decrease induced by anti-PD-1 was reversed by combination with Composition 002.

[0265] Next, CD45 + CD3 - F4 / F80 + CD206 - (M1) or CD206 +Tumor-associated macrophages (TAMs), gated at (M2), were assessed in three tumor subsets from all groups. The data, graphically displayed in Figures 16A-16B, demonstrate similar findings from our previous study. A consistent and significant increase in M1 macrophages and a decrease in M2 macrophages were again observed in both tumors and spleens caused by Composition 002 treatment, resulting in a high M1 / M2 ratio across all Composition 002 treatment groups, both in IV and IT administration groups. This ratio was not altered by PD-1 blockade and remained elevated in the combination group, resulting in more effective antitumor activity.

[0266] T cells (CD45 + CD3 + FACS analysis data for the immune checkpoint PD-1 expressed in tumor cells (gated by ) are shown in Figures 17A-17B. PD-1 expression levels on T cells were increased by Composition 002 treatment in both splenocytes and TILs, both intravenously and intratumorally, compared to the control group, but this was more pronounced and statistically significant in the spleen compared to the tumor, and was more pronounced in the intravenously delivered group. The percentage of PD-1 in the spleen or tumor of mice treated with anti-PD-1 remained significantly lower compared to the group administered Composition 002, but the decrease induced by the anti-PD-1 antibody was only statistically significant in the spleen when compared to the control group. In the combination group, PD-1 expression remained elevated compared to that measured in the single-agent groups, likely due to the augmenting effect of Composition 002.

[0267] As shown in Figures 18A-18B, intravenous delivery of Composition 002 was greater in the tumor than in the spleen, demonstrating a significant reduction in PD1 + Macrophages (CD45 + , CD3 - F4 / 80 + Anti-PD-1 antibodies increased the percentage of PD1 in the spleen (gated in ), but did not increase intratumoral delivery. +Although Composition 002 did not affect the percentage of macrophages, it statistically significantly reduced the percentage in tumors compared to control baseline in the intravenous administration group. While the effects of Composition 002 and the anti-PD-1 antibody were not consistent between the two administration groups, this was simply due to the difference in PD-1 expression in only three selected samples between the two delivery groups. + This may be due to differences in baseline levels of macrophages.

[0268] In the intravenous combination group, intratumor PD1 + Macrophages remained low and were statistically different compared to the values ​​measured upon treatment with Composition 002.

[0269] As shown in Figures 19A-19B, T cells (CD45 + CD3 + Expression of PD-L1 on tumors (gated by ) is higher in the spleens of mice treated intravenously with Composition 002 compared to controls and is not affected by the anti-PD-1 antibody. In the intratumoral administration group, a statistically significant difference is observed between the control group and anti-PD-1 treated tumors.

[0270] In the group treated with the combination of Composition 002 and anti-PD-1, PD-L1 + The percentage of T cells remained high due to the increasing effect of Composition 002, as did the results of PD-1 expression on T cells.

[0271] Finally, PD-L1 shown in Figures 20A-20B + Analysis of macrophage data shows that Composition 002 induced a consistent increase in the spleen and tumor in both the intravenous and intratumoral groups, which was unchanged by the anti-PD-1 antibody. This increase was stronger and statistically significant in the spleen than in the tumor, and was maintained upon combined treatment with Composition 002 and the anti-PD-1 antibody.

[0272] All flow cytometry analysis data above focuses on a comparison of one dose (10 mg / Kg) of Composition 002 administered intravenously or intratumorally to mice as a single agent and in combination with an anti-PD-1 antibody.

[0273] In this study, a higher dose of Composition 002 (20 mg / Kg) was also tested via the intratumoral route, and the data are shown in Figures 21A-21K. Primarily, the high dose of Composition 002 did not significantly affect the analyzed immune phenotypes, except for inducing a statistically significant slight increase (approximately 1.5-fold) in the M1 / M2 ratio in splenic T cells compared with the low dose. Furthermore, the combination of Composition 002 with anti-PD-1 at a dose of 20 mg / Kg was more effective at increasing M1 macrophages than the combination of Composition 002 with anti-PD-1 at a dose of 10 mg / Kg. Similarly, the combination of Composition 002 with anti-PD-1 at a high dose slightly increased the percentage of PD-1 in splenic T cells compared with the combination of the low dose (statistically significant relative to the control group, Group 1). No such effect was observed in tumors with either of the two doses of Composition 002 delivered intratumorally.

[0274] conclusion Findings from this study showed that single-agent administration of Composition 002 significantly inhibited EMT6 tumor growth (which replicated the results of our previous unpublished study) and that the combination of Composition 002 with an anti-PD-1 antibody provided superior anti-tumor activity.

[0275] Consistent with our previous observations, intravenous drug delivery in this study was more effective than intratumoral administration. Composition 002 administered intratumorally to mice at doses of 10 and 20 mg / kg did not produce a dose-response effect. The higher dose of 20 mg / kg was shown to be unsafe and could not be tested via the intravenous route. Therefore, unless drug penetration and distribution through tumor tissue are improved, it remains unclear whether increasing tumor exposure through a higher-dose drug regimen will result in increased efficacy.

[0276] The most significant and consistent changes induced by Composition 002 were on tumor-associated macrophages (TAMs) and PD-1 + / PD-L1 + T cells and PD1 + / PD-L1 + A higher percentage of macrophages was observed, concomitant with an increase in M1 cell types and a decrease in M2 cell types. Consistent with the paradigm that M1 represents antitumor activity and M2 promotes tumor progression, an increase in the M1 / M2 ratio explains one mechanism of antitumor activity.

[0277] As reported in the literature, the EMT6 breast tumor model shows a weak response to anti-PD-1 antibodies, and in this study it was shown to be completely resistant to this immune checkpoint inhibitor. Interestingly, despite the lack of efficacy with anti-PD-1 alone, the combination of Composition 002 with anti-PD-1 antibodies further reduced tumor growth compared to monotherapy.

[0278] The high expression of PD-1 on T cells in the treatment group is likely due to the high immune activity and favorable immune microenvironment stimulated by Composition 002. However, anti-PD-1 treatment of EMT6 tumors is not sufficient to block the immunosuppressive microenvironment; therefore, Composition 002 may be a promising drug targeting MDSCs to overcome resistance to anti-PD-1.

[0279] Furthermore, targeting PD-L1 in addition to PD-1 in combination with Composition 002 may be an even better treatment for EMT6 and ultimately triple-negative breast cancer in the clinic.

[0280] Example 2: In vivo efficacy of Streptococcus pyogenes nonviable cells in the MBT-2 syngeneic bladder cancer model subcutaneously implanted in C3H / HeN mice, as monotherapy and in combination with anti-mPD-1

[0281] The antitumor efficacy of Composition 002, a lyophilized preparation of penicillin-treated Streptococcus pyogenes (group A, type 3, substrain), alone and in combination with an antibody targeting programmed cell death protein 1 (PD-1), was evaluated in the murine bladder tumor model MBT-2 implanted subcutaneously (sc) into immunocompetent C3H / HeN mice. Efficacy studies were initiated in eight groups of 15 mice per group, who received daily sc administration of lyophilized Streptococcus pyogenes at doses of 2, 1, and 0.5 mg / kg, alone or in combination with twice-weekly intraperitoneal (ip) administration of 5 mg / kg anti-mPD-1. One group received 5 mg / kg anti-mPD-1 alone, and one group received lyophilized Streptococcus pyogenes vehicle as a reference control. Tumor volumes at study entry ranged from 50 to 150 mm. 3 The study endpoint was a tumor volume of 1,500 mm in the first animal. 3 The study was completed when the termination criterion of >100% was met. The study was terminated in two cohorts: Groups 2–7 were terminated on Day 8, and Groups 1 and 8 were terminated on Day 10.

[0282] Antitumor efficacy of all groups was assessed using the vehicle control group as a baseline. Tumor samples collected at termination were used for downstream analysis of tumor-infiltrating leukocytes (TILs). Two predefined marker panels were used to measure CD4+ in tumors from 10 animals in each group. + and CD8 +T cell, Treg, granulocytic MDSC, monocytic MDSC, NK cell, and M1 / M2 macrophage populations were assessed by FC analysis. EDTA plasma samples collected during and at the end of the study, as well as tumor samples, were analyzed to assess changes in various cytokines using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A. These data are reported separately.

[0283] In this study, Composition 002 at 0.5, 1, and 2 mg / kg in monotherapy and anti-mPD-1 at 5 mg / kg in monotherapy did not demonstrate anti-tumor activity against the MBT-2 tumor model. The combination of Composition 002 at 0.5, 1, or 2 mg / kg with anti-mPD-1 was also not effective against the MBT-2 tumor model in this study, with no statistically significant differences in tumor volume observed between any of the test groups and the vehicle control group (Kruskal-Wallis test combined with Dunn's post-hoc test).

[0284] FC analysis of cells isolated from MBT-2 tumors at the end of the study showed that tumors from all study groups were significantly more CD45+ than those from the control group. + It was shown that the percentage of cells was low. + and CD8 + The intragroup variation in cell percentages was very high. Compared to the control group, Tregs were significantly increased in the three Composition 002 monotherapy groups, the anti-mPD-1 monotherapy groups, and the 1 mg / kg Composition 002 / anti-mPD-1 group (Kruskal-Wallis test combined with Dunn's post-hoc test). Compared to the control group, the percentage of granulocytic MDSCs was significantly lower, while the percentage of monocytic MDSCs was significantly higher, in all test groups except the 2 mg / kg Composition 002 monotherapy and 0.5 mg / kg Composition 002 / anti-mPD-1 groups. No significant differences were observed between the test and control groups for NK cells. Compared to the control group, the frequency of M1 macrophages was significantly higher, while the frequency of M2 macrophages was significantly lower, in all test groups except the 0.5 mg / kg Composition 002 / anti-mPD-1 group.

[0285] In this study, after adjusting for animals excluded for tumor-related reasons, a slight decrease in group mean body weight and a survival rate of 87-100% were observed, indicating that the test substance was well tolerated.

[0286] The efficacy study consisted of eight groups of 15 mice each treated with three dose levels of Composition 002, alone or in combination with anti-mPD-1, as summarized in Table 8. Tumor volumes at the start of the study ranged from 50 to 150 mm 3 The range was. [Table 23]

[0287] The study endpoint was a tumor volume of 1,500 mm in the first animal. 3 The study was completed when the termination criterion of >100% was met. The study was terminated in two cohorts: Groups 2–7 were terminated on Day 8, and Groups 1 and 8 were terminated on Day 10.

[0288] Tumor samples collected at termination were used for downstream analysis of tumor-infiltrating leukocytes (TILs). Two predefined marker panels were used to measure CD3+ / CD3+ in tumors from 10 animals in each group. + / CD4 + and CD3 + / CD8 + T cell, Treg, granulocytic MDSC, monocytic MDSC, NK cell, and M1 / M2 macrophage populations were assessed by FC analysis.

[0289] EDTA plasma samples collected 3 days after treatment initiation and at the end of the study, as well as tumor samples, were analyzed to assess changes in various cytokines using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A.

[0290] Table 9 summarizes the samples collected in this study. [Table 24]

[0291] Terminal plasma samples were divided into 70 μl aliquots and a remainder.

[0292] The antitumor efficacy of all groups was evaluated using the vehicle control group as a reference. Tumor growth inhibition was determined by comparing the RTV of the test group with the vehicle control group and expressed as a percentage as the minimum T / C value.

[0293] A summary of the engraftment and randomization data is shown in Table 10. Individual dosing schedules, efficacy, body weight and survival data are shown in Tables 11 and 12. Tumor growth curves and FC data are shown in Figures 22-24. [Table 25] [Table 26] [Table 27-1] [Table 27-2]

[0294] material and method Test Reagents [Table 28]

[0295] handling [Table 29]

[0296] formulation Solvent for anti-mPD-1: PBS Solvent for composition 002: 0.9% NaCl Test reagents were dissolved or diluted in the appropriate solvent on the day of administration as shown in the table below. [Table 30]

[0297] All dosing solutions were administered in a dosing volume of 10 ml / kg.

[0298] animal Animals (female C3H / HeNCrl mice) were shipped from Charles River at a standard age of 4–6 weeks and allowed a minimum of 1 week of acclimation before use. At the time of tumor implantation, animals were randomly numbered using radio frequency identification transponders (Planet ID). Each cage was labeled with a record card listing all relevant study details.

[0299] Animals were housed in individually ventilated cages (TECNIPLAST Sealsafe-IVC-System, TECNIPLAST, Hohenpeissenberg, Germany) in either type III or type II long cages, depending on group size. They were maintained under an artificial photoperiod of 14L:10D. The temperature inside the cage was maintained at 22 ± 1°C, the relative humidity inside the cage was 40-70%, and the air exchange inside the cage was 60-65 times / hour. Dust-free bedding and additional nesting material consisted of aspen wood chips (ABEDD, LAB & VET Service GmbH, Vienna, Austria, product code: LTE E-001) measuring approximately 5 mm × 5 mm × 1 mm were used. Cages containing bedding and nesting material were changed weekly. Animals were fed autoclaved Teklad Global Extruded 19% Protein Rodent Diet (purchased from Envigo RMS SARL) and had free access to sterile-filtered, acidified (pH 2.5) water, which was changed twice weekly. Food and water were available ad libitum. All materials were autoclaved before use.

[0300] Nutrient-enriched water gel (DietGel Recovery purchased from ClearH2O, Maine, USA) was provided in the animal cages as needed and replaced every other day.

[0301] Tumor cell culture and transplantation The bladder tumor xenografts used in this study were derived from the commercially available cell line MBT-2.

[0302] Cells were cultured in EMEM medium (CLS #820100a) supplemented with 10% (v / v) fetal bovine serum (Sigma #F9665) and 0.05 mg / ml gentamicin (Life Technologies, Karlsruhe, Germany) in a humidified atmosphere of 5% CO at 37 °C and passaged at 40–60% CO using TrypLE Express (Thermo Fisher, #12605-010). Recipient animals were anesthetized by isoflurane inhalation and 1 × 106 Tumor cells (100 μl of PBS suspension) were administered by subcutaneous injection into the right flank. Cell viability in the cell suspension was measured before and after tumor implantation using a CASY TT cell counter (OLS OMNI Life Science GmbH & Co. KG, Bremen, Germany).

[0303] Register / Start Exam Tumor implants in a sufficient number of animals were grown to a test volume of 50–150 mm 3 Animals were monitored until a tumor volume of 1000 mg / kg / day was reached. Mice were assigned to groups so that the group median tumor volume and group mean tumor volume were equivalent. The process of group assignment (enrollment, stratified randomization) is referred to herein as randomization. The randomization day was designated as day 0 of the study.

[0304] The time from implantation to randomization at the required tumor volume is expressed in days as the "Induction time (IT)."

[0305] Animal Monitoring Animals were monitored regularly, at least twice daily on weekdays and at least once daily on weekends and holidays. Regular monitoring included identifying dead animals, observing animal welfare and tumor growth, and controlling food and water supplies and technical housing conditions. All observed or suspected animal welfare compromises were recorded. Observations and potential outcomes, such as application of euthanasia criteria or implementation of veterinary care, are presented along with study data in Table 12. Necropsies of animals were performed when deemed necessary.

[0306] body weight Animals were weighed daily for the first week, then three times a week, or daily if a weight loss of more than 10% was recorded. The relative weight of an individual animal was calculated as the individual body weight on day x (BW x ) divided by the individual's body weight on the day of randomization (BW0) and multiplied by 100:

number

[0307] Group mean relative body weight (RBW) was calculated for evaluation. Group mean RBW values ​​were reflected in the graph as long as at least 50% of the animals in the group survived.

[0308] Individual weight change % from the date of randomization x Weight change (BW x - BW0) divided by the weight on the day of randomization (BW0) and multiplied by 100.

number

[0309] Tumor volume Absolute tumor volume (ATV) was measured by two-dimensional measurements using digital calipers (SCal EVO Bluetooth, Switzerland) on the day of randomization and three times weekly thereafter. Tumor volume was calculated according to the following formula:

number

[0310] The relative volume of an individual tumor on day x (individual RTV) is calculated by dividing the absolute volume of an individual tumor on day x (T x ) divided by the individual absolute tumor volume of the same tumor on the day of randomization (T0) and multiplied by 100:

number

[0311] Group mean RTV values ​​were used for growth curve construction and treatment evaluation as long as at least 50% of the animals in the group survived.

[0312] Calculation of group mean tumor volume included values ​​from all animals surviving on that day.

[0313] Treatment implementation Dosing was performed as described in Table 11. Sc treatment was administered in a flank skinfold rather than the typically used neck skinfold to facilitate post-treatment blood sampling.

[0314] The first administration day was the day of randomization (day 0).

[0315] Dose adjustment When significant weight loss is recorded in an efficacy study, the following actions will be taken: Weigh individual animals daily with weight loss >10% No treatment is given to individual animals with weight loss >15% Facilitate free access to food and water for animals with weight loss >10% Resume administration when individual animals have recovered to RBW ≥ 90%

[0316] Note: If any animal requires easy access to food and water ad libitum, all animals in that group / cage will be offered DietGel. Other compromises of animal welfare may also warrant discontinuation of administration under the direction of the responsible veterinarian.

[0317] Euthanasia Standards In accordance with animal welfare regulations and the relevant SOPs of Charles River Discovery Research Services Germany, the following humane endpoints apply to each individual animal, regardless of study status: Tumor volume > 1500 mm 3 (Before the weekend, tumor volume > 1200 mm 3 ) ·Ulcerating skin-penetrating tumors Skin necrosis at the tumor site >5-8 mm in diameter Weight loss >30% on any measurement date Weight loss >20% sustained for more than 2 days Rapid weight loss >20% recorded within 2 days Severe impairment of the general condition (lethargy, pain, marked reduction in food and water intake, difficulty breathing, abnormal body shape or behavior)

[0318] When individual animals met the euthanasia criteria, sampling was performed prior to the scheduled time, and if possible at an appropriate time interval after administration of the last applicable dose.

[0319] Tumor samples Tumors were collected immediately after euthanasia and, if possible, divided into three portions: the first third was prepared for FC analysis, the second portion was snap-frozen in liquid nitrogen for cytokine analysis, and the third portion was transferred to fixative (FFPE sample).

[0320] Fixation was performed in 10% neutral buffered formalin for approximately 24 hours. The fixative was then replaced by soaking the samples in 70% ethanol for up to 7 days. The samples were then dehydrated by sequential incubation in the following solutions: 70% ethanol (0.5 h, twice), 80% ethanol (1 h, twice), 100% ethanol (0.5 h, twice), 100% isopropanol (1.5 h), and xylene (2 times: 1 h and 1.5 h). Finally, the samples were infiltrated and embedded in paraffin.

[0321] Tumor samples were not collected in cases of tumor remission or severe ulceration at the tumor site.

[0322] For FC analysis, tumors were cut into 2-4 mm fragments and processed with the Miltenyi Mouse Tumor Dissociation Kit according to the manufacturer's instructions. Briefly, tumor pieces were incubated with the provided enzyme mixture on a gentleMACS Dissociator, and the resulting cell suspension was filtered through a MACS SmartStrainer (100 μm; Miltenyi, # 130-110-917). The strainer was washed, and the cells were centrifuged at 300 × g for 5 min. The supernatant was discarded.

[0323] Cells were resuspended in 1x ACK lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.2-7.4) and incubated at room temperature for 1-3 minutes. Cells were pelleted by centrifugation at 300 x g for 5 minutes, and the supernatant was removed. Cells were washed by resuspending in FC buffer (2% FBS in PBS) and centrifugation at 300 x g for 5 minutes. The supernatant was removed, and cells were resuspended in FC buffer, counted, and collected at 5 x 10 per well. 5 Cells were processed for FC analysis.

[0324] blood sample Blood was collected by retrobulbar sinus puncture under isoflurane anesthesia.

[0325] Blood was collected on ice into standard plasma vials containing EDTA as an anticoagulant and plasma was prepared by immediate centrifugation twice at 2,000 × g for 5 min at 4° C. Plasma was transferred to new tubes on ice, and samples were stored at −80° C. until analysis or transport.

[0326] Flow cytometry [Table 31] [Table 32]

[0327] The flow cytometry (FC) buffer used was 2% FBS in PBS. The Fc-blocking antibody used was purified CD16 / 32 (2.4G2), 0.5 mg / ml (#553142 - BD Biosciences).

[0328] Cells were plated in a 96-well plate (5 × 10 5 Cells were then transferred to a 100µL well (100 cells / well) of PBS buffer. The cells were pelleted by centrifuging the plate at 400 x g for 5 minutes, and the supernatant was removed. An Fc-blocking antibody (10µl / well diluted 1:100 in Fc buffer) was added to each well, and the plate was incubated at room temperature for 5 minutes. Specific antibodies targeting cell surface markers (see section 5.3.10.1 for the antibodies used; antibody panels A and B were stained separately) were then added to Zombie Aqua Fixable Viability stain (diluted 1:100 in PBS buffer) as recommended by the manufacturer, and the plate was incubated at 4°C for 30 minutes, protected from light. The cells were washed by adding 200µl of Fc buffer and centrifuging the plate at 400 x g for 5 minutes, and the supernatant was removed.

[0329] To stain intracellular mouse FoxP3, 200 μl of fixative (BD Pharmingen #51 9006124) was added to the relevant wells and the plate was incubated at 4°C for 30 minutes, protected from light. The cells were pelleted by centrifugation at 400 × g for 5 minutes at room temperature, and the fixative was removed. The cells were resuspended in 200 μl of permeabilization solution (BD Pharmingen #51 9006125) prewarmed to 37°C, washed by centrifugation at 400 × g for 5 minutes, and the permeabilization solution was carefully removed. Fresh permeabilization solution (200 μl) was added to the relevant wells and the plate was incubated at 37°C for 30 minutes, protected from light. The cells were pelleted by centrifugation at 400 × g for 5 minutes, and the permeabilization solution was discarded. The cells were washed once with 200 μl of FC buffer and incubated with FoxP3 antibody in 40 μl of FC buffer per well. After 20 minutes of incubation at room temperature in the dark, 200 μl of FC buffer was added, the plate was centrifuged at 400×g for 5 minutes, and the FC buffer was removed.

[0330] Finally, the cells were resuspended in 200 μl of FC buffer, transferred to a deep-well plate containing 200 μl of FC buffer, and analyzed using an Attune NXT Acoustic Focusing Cytometer (Violet (405 nm) / Blue (488 nm) / Yellow (561 nm) / Red (638 nm) laser configuration).

[0331] Data evaluation survival rate The survival rate (Table 12) was calculated by counting the number of animals in each group that would have survived beyond the final study day and dividing it by the total number of animals in that group. Animals that died or were euthanized on the final day of the group for reasons other than sample collection or group termination were not counted as survivors. The adjusted survival rate in Table 2 was calculated by counting all survivors, including animals euthanized for tumor-related reasons, and dividing it by the total number of animals in that group. The following reasons for euthanasia are classified as tumor-related: 1) tumors that meet the volume-related euthanasia criteria, including secondary tumors, and 2) ulcerated tumors. Euthanasia of animals due to symptoms of tumor-induced cachexia is not counted as tumor-related.

[0332] Tumor volume doubling time / quadrupling time The tumor volume doubling time and quadrupling time (Td, Tq) of the test and control groups are defined as the time interval (in days) required for the group to reach a median RTV of 200% or 400%. The data are shown in Table 11.

[0333] Tumor growth inhibition, test / control value (%) (minimum T / C value) The values ​​of the test group vs. the control group on a particular day (T / C 平均 The percentage (unit: %) was calculated by multiplying the ratio of the mean RTV values ​​of the test group and the control group on day x by 100.

number

[0334] The minimum (or optimal) T / C recorded in the test group during the study 平均 Values ​​represent the maximum antitumor efficacy for each treatment. 平均 Note that values ​​were calculated including values ​​obtained using the LOCF method.

[0335] Group minimum T / C 平均 The values ​​were used for efficacy evaluation as follows: [Table 33]

[0336] Flow cytometry analysis Flow cytometry data were analyzed using FlowJo data analysis software. The software automatically determined the frequency of subpopulations as a percentage of the parent population. FC results were presented as a percentage of the corresponding parent population and the total number of each population. Doublet exclusion was performed based on forward scatter height versus forward scatter area to include only single cells, followed by leukocyte gating and live / dead cell discrimination determined by forward / side scatter. Fluorescence Minus One (FMO) controls were used to establish correct gating. Further gating was performed as needed to evaluate designated populations. Information on the analyzed populations is shown in the table below. [Table 34] [Table 35]

[0337] statistical analysis For the assessment of statistical significance of antitumor efficacy, the nonparametric Kruskal-Wallis test [1] was performed, followed by the Dunn method for multiple comparisons [2].

[0338] Individual ATVs of test and control groups were compared on the final day of the study, when all groups were active. Statistical analysis was performed only when at least 50% of the originally randomized animals remained in the relevant group. No statistically significant differences in tumor volume were observed between the control and test groups.

[0339] For the FC data, Kruskal-Wallis test / Dunn's post-hoc test was performed for various cell populations to compare the percentages between the test and control groups. Statistically significant differences between the test and control groups are shown in Figures 23A-23B.

[0340] All p values ​​<0.05 were considered statistically significant. Statistical calculations were performed using R (version 3.1.0; https: / / www.r-project.org / ), in which Kruskal-Wallis tests and Dunn's post hoc tests were performed according to Hollander and Wolfe [3] or GraphPad Prism bioanalytic software (version 9.0 for Microsoft Windows, GraphPad Software, San Diego, CA, USA, https: / / www.graphpad.com / ).

[0341] Results and Discussion Antitumor efficacy In this study, the anti-tumor efficacy of Composition 002 and anti-mPD-1 was evaluated in a syngeneic MBT-2 tumor model implanted in C3H mice. A summary of the study is shown in Table 10. Efficacy results are summarized in Table 11 and Figures 22A-22B.

[0342] In this study, Composition 002 monotherapy at 0.5, 1 and 2 mg / kg did not exhibit antitumor activity against the MBT-2 tumor model (all minimum T / C values ​​= 100%).

[0343] In this study, anti-mPD-1 monotherapy at 5 mg / kg did not show antitumor activity against the MBT-2 tumor model (minimum T / C value 100%).

[0344] In this study, combination therapy of 0.5, 1, or 2 mg / kg Composition 002 with anti-mPD-1 was also ineffective against the MBT-2 tumor model (minimum T / C value ≧71.6%).

[0345] On study day 7, the final day all groups were on study, no statistically significant differences in tumor volume were observed between the test and vehicle control groups.

[0346] Flow cytometry analysis FC analysis was performed on cells isolated from MBT-2 tumors at the final time point, day 10 (groups 1 and 8) or day 8 (groups 2-7). FC results are shown in Figures 23A-23F (percentage) and Figures 24A-24F (cell count). Key findings are as follows:

[0347] CD45 + The percentage of cells was lower in tumors from all test groups compared to the control group (Figure 23A). When this difference was analyzed with Panel A antibodies, it was statistically significant in the 2 mg / kg Composition 002 / anti-mPD-1 and 1 mg / kg Composition 002 / anti-mPD-1 groups. Analysis with Panel B antibodies yielded similar results, but also showed significant differences in the 1 mg / kg and 0.5 mg Composition 002 monotherapy groups.

[0348] CD3 + CD11b - The frequency of T cell populations was significantly elevated in all test groups except for the 0.5 mg / kg Composition 002 / anti-mPD-1 group compared to the control group. + and CD8 + The intragroup variation in cell percentage was very large, with the only significant difference observed being the CD4 + Only Treg cells were detected, and their frequency was statistically significantly lower compared to the control group. Tregs were significantly increased in the three Composition 002 monotherapy groups, the anti-mPD-1 monotherapy group, and the 1 mg / kg Composition 002 / anti-mPD-1 group (Figure 23B).

[0349] The percentage of granulocytic MDSCs was significantly lower in all test groups except the 2 mg / kg Composition 002 monotherapy group and the 0.5 mg / kg Composition 002 / anti-mPD-1 group, while the percentage of monocytic MDSCs was significantly higher in all test groups except the 2 mg / kg Composition 002 monotherapy group and the 0.5 mg / kg Composition 002 / anti-mPD-1 group (Figure 23C).

[0350] No significant differences were observed between the test and control groups for NK cells (Figure 23E). The intragroup variability was higher in the test group than in the control group.

[0351] F4 / 80 + The frequency of M1 macrophages in the cells was significantly higher in all test groups except for the 0.5 mg / kg Composition 002 / anti-mPD-1 group, while the frequency of F4 / 80 + The frequency of M2 macrophages among the cells was significantly lower in all test groups except for the 0.5 mg / kg Composition 002 / anti-mPD-1 group (Figure 23F).

[0352] Weight change, survival and observations The results are summarized in Table 12 and Figure 25.

[0353] Minimal body weight loss (BWL) was observed in this study, indicating good tolerability of the test substance. The highest group mean BWL of 3.9% was observed in the 0.5 mg / kg Composition 002 monotherapy group, compared to 0.9% in the control group. Two animals in the 0.5 mg / kg Composition 002 monotherapy group were found dead on day 8, and two animals in the 0.5 mg / kg Composition 002 / anti-mPD-1 group were found dead on days 7 and 10, resulting in an 87% survival rate after adjusting for animals euthanized for tumor-related reasons. In the other two combination therapy groups, the 2 mg / kg Composition 002 / anti-mPD-1 group and the 1 mg / kg Composition 002 / anti-mPD-1 group, one animal was found dead on days 6 and 5, respectively, resulting in an adjusted survival rate of 93%. In all remaining groups, survival was 100%.

[0354] References [1] Kruskal WH, Wallis WA: Use of Ranks in One-criterion Variance Analysis. J. Am. Stat. Assoc. 1952, 47: 583-621. [2] Dunn OJ: Multiple Comparisons Using Rank Sums. Technometrics, 1964, 6(3), pp. 241-252. [3] Hollander M, Wolfe DA: Nonparametric Statistical Methods. New York: John Wiley & Sons, 1973, Pages 115-120.

[0355] Acronyms and abbreviations: [Table 36]

[0356] Example 3: Preparation of a composition containing lyophilized nonviable Streptococcus pyogenes for injection

[0357] Streptococcus pyogenes (group A, type 3, strain Su) is cultured in an appropriate culture medium. After an appropriate incubation period, the bacteria are harvested by centrifugation, washed, resuspended, and treated with hydrogen peroxide to kill the bacteria. The killed bacteria are centrifuged, washed, resuspended in a suspension medium such as Berheimer's basal medium (BBM), and filtered. The bacterial suspension is treated with benzylpenicillin and heated at 37°C for approximately 10-45 minutes and at 45°C for approximately 20-60 minutes. A final bulk suspension is prepared. The final bulk suspension is filled into vials and lyophilized. Quantitative compositions for various proposed dosage strengths of exemplary compositions containing nonviable Streptococcus pyogenes are shown in Table 13A. These compositions are based on lyophilized formulations. All vial strengths are filled with the same volume (0.41 mL) of suspension prior to lyophilization. [Table 37]

[0358] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005-0.01 mg / mL. The volume of product delivered to a subject at this concentration may vary. [Table 38]

[0359] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005-0.01 mg / mL. The volume of product delivered to a subject at this concentration may vary.

[0360] Example 4: T cell activation by a composition containing nonviable Streptococcus pyogenes

[0361] T cells were isolated from peripheral blood mononuclear cells (PBMCs—two healthy donors) using RapidSheres magnetic beads. T cells (500,000) were seeded into 96-well plates. T cells were treated with 0.2 and 0.8 KE / mL of Composition 002 for 72 hours. T cells and supernatants were collected for analysis of immune checkpoint biomarkers and cytokines (see table below) by FACS and ELISA, respectively. [Table 39]

[0362] As shown in Figure 26, Composition 002 treatment does not alter the number of CD4+ and CD8+ T cells. Composition 002 induces the expression of immune checkpoint molecules CTLA4, PD-1, LAG3, TIM3, and TIGIT in CD4+ T cells (Figure 27A) and CD8+ T cells (Figure 27B).

[0363] Example 5: In vivo efficacy of Streptococcus pyogenes nonviable cells in an orthotopic bladder model (MB49 bladder cancer cells) as monotherapy and in combination with anti-mPD-1

[0364] The antitumor efficacy of a lyophilized preparation of penicillin-treated Streptococcus pyogenes (group A, type 3, substrain) is evaluated in a mouse orthotopic bladder tumor model (MB49 bladder cancer cells) alone and in combination with an antibody targeting programmed cell death protein 1 (PD-1). The monotherapy study design is shown in Table 6 below. Animals are randomized into study groups based on tumor-associated bioluminescence. The lyophilized preparation of the penicillin-treated Streptococcus pyogenes composition is administered once weekly for four weeks. Mice are observed for one week post-treatment. Mice are examined daily, five days per week. Body weights are measured twice weekly. Bioluminescence imaging (BLI) is performed once or twice weekly for four weeks during the survival period. The optimal dose of the lyophilized preparation of penicillin-treated Streptococcus pyogenes for combination testing with anti-PD-1 antibodies is selected. [Table 40]

[0365] The study design for combination therapy is shown in Table 15 below. Animals are randomized into study groups by tumor-associated bioluminescence. A lyophilized preparation of penicillin-treated S. pyogenes composition is administered once a week for four weeks. Anti-PD-1 antibody is administered twice a week. Mice are observed for one week after treatment. Mice are examined daily, five days a week. Body weights are measured twice a week. Bioluminescence imaging (BLI) is performed once or twice a week for four weeks during survival. [Table 41]

[0366] Example 6: Preparation of a composition containing lyophilized nonviable Streptococcus pyogenes for injection

[0367] Streptococcus pyogenes (group A, type 3, strain Su) is cultured in an appropriate culture medium. After an appropriate incubation period, the bacteria are harvested by centrifugation, washed, resuspended, and treated with hydrogen peroxide to kill the bacteria. The killed bacteria are centrifuged, washed, resuspended in a suspension medium such as Berheimer's basal medium (BBM), and filtered. The bacterial suspension is treated with benzylpenicillin and heated at 37°C for approximately 10-45 minutes and at 45°C for approximately 20-60 minutes. A final bulk suspension is prepared. The final bulk suspension is filled into vials and lyophilized. The quantitative compositions for various proposed dosage strengths of exemplary compositions containing nonviable Streptococcus pyogenes are shown in Table 16. These compositions are based on the lyophilized formulation. All vial strengths are filled with the same volume (0.41 mL) of suspension prior to lyophilization. [Table 42]

[0368] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension with a concentration of 0.005-0.01 mg / mL. The volume of product delivered to a subject at this concentration may vary. Table 17 provides the quantitative composition of an exemplary composition suspended in 0.9% saline at a final cell concentration of 0.01 mg / mL. [Table 43]

[0369] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005-0.01 mg / mL. The volume of product delivered to a subject at this concentration may vary.

[0370] Example 7: In vitro and in vivo efficacy of nonviable Streptococcus pyogenes cells in monotherapy and in combination with immune checkpoint inhibitors

[0371] material and method: immunogenic cell death To assess the release of damage-associated molecular pattern molecules (DAMPs), bladder tumor MB49 cells were plated in triplicate at each test point in 96-well plates (2 × 10 4 Cells were seeded onto the plate (1000 x 1000 cells / well) and incubated for 24 hours at 37°C in 5% CO2 in DMEM high glucose phenol red-free medium containing HEPES (Thermostat, Cat. No. 21063029), 10% heat inactivated fetal bovine serum (HI FBS) (Seradigm Avantor, Cat. No. 1500-500H), and 1% penicillin / streptomycin (P / S) (Gibco-Thermofisher, Cat. No. 15140-122).

[0372] Cells were treated with 0, 0.2, 0.8, 3.2, or 12.8 KE / mL (1 KE = 0.1 mg) of Composition 002 or 1 μM mitoxantrone (Sigma, Cat. No. M6545) as a positive control for 24 hours. After treatment, the plates were centrifuged at 400 g for 5 minutes, the supernatant was collected for HMGB1 quantification, and the cells were collected for flow cytometry analysis.

[0373] HMGB1 was quantified using the Promega Lumit Immunoassay (Promega, Cat. No. W6110) kit according to the manufacturer's instructions. The mean value of the appropriate background control RLU (medium alone or medium alone treated with drug at the corresponding concentration) was subtracted from each triplicate measurement of the treated cell sample data. The fold induction of the treated sample was calculated according to the following formula:

number

[0374] Annexin V-FITC (Abcam, Cat. No. Ab14085) and calreticulin-AF700 (R&D Systems, Cat. No. IC38981N) markers were quantified by flow cytometry. Briefly, cells were stained with calreticulin-AF700 for 30 minutes at 4°C, washed, and resuspended in 1x binding buffer from the Annexin V kit (Abcam Cat. No. Ab14085). Annexin V and 50 μg / mL propidium iodide (Abcam Cat. No. Ab14085) were added to the solution and incubated in the dark at 25°C for 5 minutes.

[0375] For extracellular ATP (eATP) analysis, RealTimeGlo eATP Assay Reagent (Promega, Cat. No. GA5010) was added prior to Composition 002 treatment, and eATP luminescence measurements were performed 24 hours prior to treatment. After the final measurement, 400 μg / mL of Digitonin (Promega, Cat. No. G9441) was added to the medium to assess total ATP and general cell health as an assay control. eATP was calculated according to the formula:

number

[0376] Dendritic cell activation and phagocytosis assays Bone marrow cells were collected by flushing the femurs with RPMI 1640 medium (ATCC, Cat. No. 30-2001). Disaggregated cells were filtered twice through a 70 μM pre-wetted filter to remove cell clumps and counted using methylene blue in 3% acetic acid (StemCell Technologies, Cat. No. 07060). The bone marrow cells were then resuspended in RPMI 1640 medium (ThermoFisher, Cat. No. 30-2001) supplemented with 2 mM GlutaMax, 10% HI FBS (Avantor, Cat. No. 1500-500H), 1% P / S (Gibco, Cat. No. 15140-122), 50 ng / ml GM-CSF (PeproTech, Cat. No. 300-03), and 25 ng / ml IL-4 (PrepoTech, Cat. No. 200-04) and plated in a 96-well plate (3 × 10 4 The cells were cultured at 1000 x g (1000 x 1000 cells / well) at 37°C and 5% CO for 48 hours. Dendritic cell (DC) differentiation was continued for 24 hours after half of the medium was replaced with a fresh medium. The medium was then completely replaced with a fresh medium, and the cells were cultured for an additional 72 hours to complete DC differentiation.

[0377] MB49 cells (2.5×10 6 Cells) were cultured in T-25 flasks in DMEM high glucose medium containing HEPES (ThermoFisher, Cat. No. 12430054), 10% HI FBS, and 1% P / S and incubated with Composition 002 (0, 0.05, 0.2, 0.8, 1.6, and 3.2 KE / mL) for 24 hours. Dinaciclib (1 μM) (Tocris R&D, Cat. No. 7336) was used as a positive control. After treatment, cells were resuspended and washed to remove Composition 002 and then resuspended in Vybrant HCl. TM The cells were stained with DiO Cell-Labeling Solution (ThermoFisher, Cat. No. V22886) at 37°C for 20 minutes.

[0378] Pre-labeled MB49 cells and DCs were mixed in a 96-well plate at a 2:1 ratio (3 × 104 DC of pieces: 1.5×10 4 DCs were identified using CD80-PE (BioLegend, Cat. No. 305207), CD86-BV421 (BioLegend, Cat. No. 305425), CD11c-AP (BioLegend, Cat. No. 337207), and HLA-DR-BUV395 (BD Bioscience, Cat. No. 565972). Pre-labeled MB49 cells were identified using DiO-FITC. Total cell viability was analyzed by flow cytometry using APC-eFluor780 (eBioscience, Cat. No. 65-0865-14).

[0379] Cytotoxicity and Cytokine Release of Composition 002 Bladder cancer RT112 cells were cultured in EMEM medium (ATCC, Cat. No. 30-2003) supplemented with 2 mM GlutaMax (ThermoFisher, Cat. No. 35050061), 1% NEAA (ThermoFisher, Cat. No. 11140050), 10% HI FBS, and 1% P / S. 5637 cells were cultured in RPMI 1640 medium (ATCC, Cat. No. 30-2001) supplemented with 2 mM Glutamax, 10% HI FBS, and 1% P / S. Both cell lines were prelabeled with CellTracker Red solution (1 μM) (Invitrogen, Cat. No. C34552) at 37°C for 30 minutes. Cells were then washed and plated in a 96-well plate (2.5 × 10 4 Cells / well were seeded and incubated at 37°C, 5% CO2 for 24 hours.

[0380] Fresh peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using the EasySep Direct Human PBMC Isolation Kit (Stem Cell Technologies, Cat. No. 19654) according to the manufacturer's instructions and supplemented with 6 mM EDTA (ThermoFisher, Cat. No. 15575-038). PBMCs were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P / S.

[0381] Composition 002 treatment (0.2 KE / mL) was administered to tumor cells alone (2.5 × 10 4 cells / well) or PBMCs (1.65 × 10 5 The cells were co-cultured with 1.25 μg / mL anti-CD3 (eBioscience, Cat. No. 16-0037) at an effector:target cell ratio of 6.6:1 for 72 hours at 37°C and 5% CO2. After treatment, the plates were centrifuged at 400g for 5 minutes, and the supernatant was collected. Proinflammatory cytokines were stained using V-Plex Proinflammatory Panel 1 Human (Meso Scale, Cat. No. K15049D-1) and detected using a plate reader. The cells were then washed, trypsinized, and subjected to flow cytometry analysis. Tumor cell viability was quantified using CellTracker Red-PE (Invitrogen, Cat. No. C34552), Live / Dead Dye-efluor780 (eBioscience, Cat. No. 65-0865-14), and CD45-BUV395 (BD, Cat. No. 563792); PBMCs were excluded from the analysis.

[0382] T cell activation Frozen PBMCs were thawed, and the cells were gently dissociated by resuspending them in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S containing 100 μg / mL DNase I (StemCell Technologies, Cat. No. 17951) and incubating at 25°C for 15 minutes. EasySep buffer (StemCell Technologies, Cat. No. 20144) was added to the mixture, and the cells were filtered through a 37 μM cell strainer (StemCell Technologies, Cat. No. 07900). T cells were isolated using the EasySep Human T Cell Isolation Kit (StemCell, Cat. No. 17951) according to the manufacturer's instructions.

[0383] Cells were plated in a 96-well plate (5 × 10 5 / well) and treated with Composition 002 (0.2 KE / mL) for 72 hours at 37°C, 5% CO2. The plates were centrifuged, the supernatant was collected, and IFN-g (Invitrogen, Cat. No. BMS228) and Granzyme B (Invitrogen, Cat. No. BMS2027-2) were quantified by ELISA. T cells were harvested and stained for the following markers: CD4-BUV395 (BD, Cat. No. 564724), FoxP3-PE (BD, Cat. No. 560852); CD8-FITC (BioLegend Cat. No. 344704), PD-1-PE-Cy7 (BioLegend, Cat. No. 329918), CTLA-4-BV421 (BioLegend, Cat. No. 369606), TIGIT-BV711 (BioLegend Cat. No. 372742), TIM3-BV605 (BioLegend Cat. No. 345018), LAG3-APC (BioLegend Cat. No. 369212), Ki67-efluor506 (eBioscience Cat. No. 69-5698-82), Live / Dead dye-efluor708 (eBioscience, Cat. No. 65-0865-14). Data were acquired by flow cytometry.

[0384] PD-L1 analysis in tumor cells Bladder cancer 5637 cells, cultured in RPMI-1640 medium supplemented with 2 mM Glutamax, 10% HI FBS, and 1% P / S, were pre-labeled with Cell Tracker Red and plated at 2.5 × 10 cells / well in a 96-well plate. 4 Cells / well were seeded and grown overnight.

[0385] Frozen PBMCs were thawed, and the cells were gently dissociated by resuspending them in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S containing 100 μg / mL DNase I and incubating at 25°C for 15 minutes. EasySep buffer was added to the mixture, and the cells were filtered through a 37 μM cell strainer. T cells were isolated using the EasySep Human T Cell Isolation Kit (StemCell, Cat. No. 17951) according to the manufacturer's instructions and maintained in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S.

[0386] T cells were cultured at a 6.6:1 ratio (1.65 × 10 5 Media for 5637 cells:T cells was added to the appropriate co-culture wells at a 1:1 ratio according to the cells (cells / well). 5637 cells alone and in co-culture with T cells were treated with Composition 002 (0.2 and 0.8 KE / mL) for 72 hours at 37°C, 5% CO2. After treatment, the supernatant was washed, and PD-L1 (BioLegend, Cat. No. 374510) marker was analyzed by flow cytometry in pre-labeled tumor cells.

[0387] In vitro cytotoxicity analysis using the xCELLigence Real-Time Cell Analyzer (RTCA) Fresh peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using a 2x EasySep Direct Human PBMC Isolation Kit (Stem Cell Technologies, Cat. No. 19654) according to the manufacturer's instructions and supplemented with 6 mM EDTA (ThermoFisher, Cat. No. 15575-038). PBMCs were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P / S. 5637 cells, cultured in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S, were plated in a 96-well E-Plate (Agilent, Cat. No. 300600910) at 5 x 10 per well. 5The tumor cells were seeded onto tumor-specific target cells (human PBMCs; effector / target cell ratio 6.6:1) and allowed to settle for 30 minutes to allow cell attachment. Approximately 78 hours later, effector cells (human PBMCs; effector / target cell ratio 6.6:1) and treatment were added. Composition 002 treatment (0.8 KE / mL) was administered in a co-culture system (tumor cells + PBMCs) for approximately 65 hours, either alone or in combination with the following antibodies: anti-PD-1 (10 μg / mL - Bioxcell, Cat. No. SIM0010), anti-PD-L1 (10 μg / mL - Bioxcell, Cat. No. SIM0009), anti-CTLA-4 (10 μg / mL - Selleckchem, Cat. No. A20001). RecombiMAb IgG4 (Bioxcell, Cat. No. CP147) (10 μg / mL) and RecombiMAb IgG1 (Bioxcell, Cat. No. CP147) (10 μg / mL) were used as isotype controls. Cell Index measurements were collected every 15 minutes using an xCelligence RTCA eSight (Agilent) for a total of 143.5 hours (approximately 78 hours for cancer cells alone + ~65 hours for co-culture). For each well, % cell lysis was calculated using the normalized sample cell index and the normalized average target-only control according to the following formula:

number

[0388] MB49 subcutaneous mouse model For the dose-finding study, 14-week-old female C57BL / 6 mice were inoculated with MB49 bladder cancer cells (2.0 x 10 5細胞 Each group consisted of 10 mice per group, and 8 days later, the tumor size for enrollment was 80–130 mm. 3 Mice were individually administered Composition 002 intravenously (0.08, 0.4, and 2 KE / mouse) once a week for 4 weeks. Tumor measurements were performed twice a week using calipers. Humane endpoints (body weight loss >20%, tumor burden >2000 mm) were achieved. 3Animals that did not reach clinical endpoint (open exudative tumor ulceration, severe respiratory distress, severe motor impairment, or loss of righting reflex) were monitored for up to 35 days.

[0389] For the combination study, 14-week-old female mice were inoculated with MB49 cells (2.0x10 5細胞 Each group consisted of 10 mice per group, and 6 days later, the tumor size for enrollment was 75–130 mm. 3 Mice were intravenously administered Composition 002 at 0.4 KE / mouse once a week for 4 weeks and / or anti-PD-1 (10 mg / Kg) twice a week for 2 weeks (BioXcell, Cat. No. BP0146). An isotype control (2A3) was used as a negative control (Bioxcell, Cat. No. BP0089). Animals that did not reach the humane endpoint were maintained for 5 days after the final dose of Composition 002, euthanized, and tumors were collected and processed for immunohistochemistry analysis.

[0390] EMT6 orthotopic mouse model. For the dose-finding study, 5 x 10 5 EMT6 triple-negative breast cancer cells were implanted. Seven days later, six mice per group were randomized (tumor size for enrollment was 50–150 mm). 3 The tumors were 0.4, 1, and 2 KE / mouse), and Composition 002 was administered intravenously twice weekly (0.4, 1, and 2 KE / mouse). Tumor growth and mouse body weights were measured twice weekly for 3 weeks. Mice that did not reach the humane endpoint were euthanized on study day 35. Tumors from 3 mice per group were harvested and chemically dissociated by mechanical dissociation followed by treatment with collagenase D (2.5 mg / mL). Cells were filtered through a 70 μm cell strainer and stained for FACS analysis of tumor-infiltrating immune cell populations (Table 18). [Table 44]

[0391] result: MB49 bladder cancer cells exposed to Composition 002 underwent apoptosis (Figure 28A) with the production of products characteristic of immunogenic cell death, such as HMGB1, extracellular ATP (eATP), and expression of calreticulin on the cell surface (Figures 28B-28D). These damage-associated molecular patterns (DAMPs) serve as signals to attract and activate antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), which can effectively activate naive T cells. Because DCs play a key role in the recognition of DAMPs associated with immunogenic cell death and the subsequent uptake and presentation of tumor antigens, we examined the phagocytosis of Composition 002-treated tumor cells by DCs. MB49 cells were treated with Composition 002 and then cocultured with murine myeloid-derived DCs. Composition 002-treated MB49 bladder cancer cells exhibited increased dendritic cell phagocytosis (Figure 29A) and enhanced phenotypic maturation, as indicated by upregulated cell surface expression of CD80, CD86, and HLA-DR (Figures 29B-29D). Using an in vitro coculture approach of bladder cancer cells (5637 and RT112) with PBMCs, Composition 002 was found to enhance immune-mediated bladder cancer cell killing (Figures 30A-30B). Notably, cytokine analysis indicated that Composition 002 increased the release of pro-inflammatory Th1 cytokines (Figure 31A), creating a favorable environment for the induction of cellular and humoral antitumor immunity. Furthermore, Composition 002 also promoted a reduction in Th2 cytokines, which correlate with tumor growth (Figure 31B). For example, Composition 002 reduced IL-10 release, which is known to inhibit the secretion of various Th1 cytokines by macrophages and dendritic cells.

[0392] To determine whether Composition 002 induces PD-L1 expression in cancer cells and thereby suppresses antitumor immune responses, bladder cancer cells (5637) cocultured with PBMCs were treated with increasing concentrations of Composition 002. Composition 002 treatment resulted in increased PD-L1 expression in 5637 cells compared to controls (Figure 34). Furthermore, Composition 002 stimulation of T cells alone increased T cell proliferation, as observed by a higher percentage of CD4 and CD8 cells expressing the KI67 marker (Figures 32A and 32H). In addition, Composition 002 promoted IFN-γ and granzyme B release (Figures 33A-33B), suggesting enhanced cytotoxic activity. However, exhausted T cell phenotype markers were identified by high levels of LAG3, CTLA4, PD-1, TIGIT, and TIM3 in CD4 and CD8 T cells (Figures 32B, 32C, 32D, 32E, 32F, 32J, 32I, 32K, 32L, and 32M, respectively) and FOXP3 CD4+ regulatory T cells (Figure 32G). Taken together, the immunostimulatory effects of Composition 002 can be enhanced by combining it with additional agents that target these exhausted T cell phenotypes.

[0393] To investigate the synergistic antitumor effects of Composition 002 with the immune checkpoint inhibitors (ICIs) anti-PD-L1, anti-CTLA4, and anti-PD1 in vitro, we selected the xCELLigence platform, which can be used to monitor cell health, proliferation, and cytolysis over time. Target 5637 bladder cancer cells were seeded onto biosensor plates (E-Plates) and allowed to attach and grow. After 78 hours, fresh PBMCs were added to 5637 cells in the presence or absence of Composition 002, ICI, or irrelevant IgG isotypes used as controls (IgG4 for anti-PD1 and IgG1 for anti-PD-L1 and anti-CTLA4) for approximately 65 hours (143.5 hours total). The combination of Composition 002 with anti-PD-L1 or anti-CTLA4 demonstrated enhanced cytolysis of 5637 cells in the presence of human PBMCs compared to the individual treatments (Figures 35A-35D). It is worth noting that the combination of Composition 002 with ICI delayed the anti-tumor effect compared with Composition 002 alone, suggesting that the combined activity was dependent on the expression of immune checkpoints (e.g., PD-L1, CTLA4) mediated by Composition 002. However, only a slight increase in cytolysis was observed when anti-PD1 antibody was used compared with Composition 002 alone (Figures 35E-35F).

[0394] Because tumor response to immunotherapies such as anti-PD-1 may depend on the interaction of several cell types in the in vivo tumor microenvironment, the combination study of Composition 002 with anti-PD-1 was repeated using a mouse model of bladder cancer. Instead of the previously used MBT2 model (Example 2), we selected the MB49 mouse bladder cancer model, which may respond to immune stimulation and is widely used in bladder cancer immunotherapy research. Furthermore, based on the observed delayed antitumor effect of the combination of Composition 002 with other ICIs in vitro, mice were treated for 3 weeks (instead of 1 week in the previous in vivo MBT2 bladder cancer study in Example 2). Composition 002 as monotherapy was effective in reducing tumor growth and prolonging survival in the MB49 subcutaneous model (Figures 36A-36B). Further analysis using Composition 002 in combination with anti-PD-1 in this same model revealed a reduction in tumor volume compared to either Composition 002 or anti-PD-1 as monotherapy (Figures 37A-37E). For example, the mean tumor volume at day 20 was 264 mm in the anti-PD1 alone and combination groups, respectively. 3 and 159 mm 3 , demonstrating additive efficacy. Furthermore, 30% complete regressions and 10% tumor-free survivors were observed in the combination treatment group. Neither complete regressions nor tumor-free survivors were observed in the monotherapy group.

[0395] The antitumor efficacy of Composition 002 was also tested in vivo using a mouse orthotopic triple-negative breast cancer (TNBC) model (EMT6 cells), which is characterized by an immunosuppressive tumor microenvironment and resistance to anti-PD1 treatment. Results showed significant, but modest, antitumor efficacy with intravenous administration of Composition 002 as monotherapy (Figure 38A). Flow cytometry analysis of immune cells identified tumor-associated macrophages (TAMs) and demonstrated an increase in antitumor macrophage 1 (M1) cell type and a decrease in tumor-promoting macrophage 2 (M2) cell type (i.e., M1 polarization), which was associated with increased PD-1 expression on T cells (Figure 38B). Systemic delivery of the combination of Composition 002 and anti-PD1 in the EMT6 TNBC mouse model demonstrated significantly superior antitumor activity compared with each agent as monotherapy, demonstrating synergistic activity between Composition 002 and anti-PD1 (Figures 39A-39B). Furthermore, combined treatment with Composition 002 and anti-PD1 reconstituted local immune cell populations by restoring PD-1 levels on T cells, increasing the levels of intratumoral CD4 T cells, promoting macrophage polarization toward an M1 phenotype (anti-tumor), and downregulating tumor-promoting Treg cells (Figures 40A-40C). Of particular importance is the fact that the combination of Composition 002 and anti-PD1 significantly increased the levels of tumor-infiltrating natural killer (NK) cells compared with the monotherapy group (Figure 40D). Indeed, researchers have highlighted the benefit of anti-PD-1 / PD-L1 therapy in improving NK cell function, finding that disrupting PD-1 / PD-L1 interaction can enhance the killing effect of NK cells against tumor cells. Furthermore, PD-1 / PD-L1 antibodies are completely ineffective in some NK-deficient mouse models.

[0396] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or set forth in the Application Data Sheets, including but not limited to U.S. Provisional Patent Application No. 63 / 479,170, filed January 9, 2023, U.S. Provisional Patent Application No. 63 / 487,224, filed February 27, 2023, and U.S. Provisional Patent Application No. 63 / 487,232, filed February 27, 2023, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to provide further embodiments using concepts from the various patents, applications, and publications.

[0397] These and other changes can be made to the embodiments in light of the above detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments described in the specification and claims, but rather to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. 1. A method for treating triple-negative breast cancer in a subject, comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

2. 10. The method of claim 1, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered intratumorally, intravenously, intramuscularly, subcutaneously, or intraperitoneally.

3. 3. The method of claim 1 or 2, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of about 10 KE or greater.

4. 4. The method of claim 3, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of at least 20 KE.

5. 4. The method of claim 3, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of at least 40 KE.

6. 6. The method of any one of claims 1-5, wherein the nonviable cells of Streptococcus pyogenes are administered to the subject at a dose of about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1 KE, about 0.5 KE, about 1 KE, about 2.5 KE, about 5 KE, about 10 KE, about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, about 60 KE, about 70 KE, about 80 KE, about 90 KE, or about 100 KE.

7. 7. The method of any one of claims 1 to 6, wherein the non-viable cells of Streptococcus pyogenes comprise cells of the Su strain of Streptococcus pyogenes.

8. 8. The method of any one of claims 1 to 7, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof, a vaccine, a nucleic acid molecule (including an inhibitory nucleic acid molecule), a gene editing system, or a small molecule.

9. 9. The method of any one of claims 1 to 8, wherein the immune checkpoint inhibitor is an inhibitor of the PD 1 / PD-L1 / PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

10. 10. The method of claim 9, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-1 inhibitor, optionally wherein the PD-1 inhibitor is an antibody.

11. 11. The method of claim 10, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrelimab, cemiplimab, sasanlimab, nofazinlimab, geptanolimab, zimvelerimab, serplulimab, pucotenlimab, prorugolimab, camrelizumab, kadnilimab, dostallimab, penprimimab, toripalimab, tislelizumab, sintilimab, or dostallimab.

12. 10. The method of claim 9, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-L1 inhibitor, optionally wherein the PD-L1 inhibitor is an antibody.

13. 13. The method of claim 12, wherein the PD-L1 antibody comprises atezolizumab, durvalumab, embafolimab, sugemalimab, cosibelimab, socazolimab, tagitanlimab, betifisolimab, resabelimab, pakmilirumab, or avelumab.

14. 10. The method of claim 9, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-L2 inhibitor, optionally wherein the PD-L2 inhibitor is an antibody.

15. 10. The method of claim 9, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-1 inhibitor and a PD-L1 inhibitor, optionally wherein the PD-1 inhibitor and / or the PD-L1 inhibitor is an antibody.

16. 10. The method of claim 9, wherein the immune checkpoint inhibitor comprises a CTLA-4 inhibitor, optionally wherein the CTLA-4 inhibitor is an antibody.

17. 17. The method of claim 16, wherein the CTLA-4 antibody comprises ipilimumab, tremelimumab, or tuvonralimab.

18. 18. The method of any one of claims 1 to 17, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered at least one day before the immune checkpoint inhibitor.

19. 19. The method of any one of claims 1 to 18, further comprising administering to the subject an additional anti-cancer agent.

20. 20. The method of any one of claims 1 to 19, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered prior to, concurrently with, or subsequent to the immune checkpoint inhibitor.

21. 21. The method of any one of claims 1 to 20, wherein the composition comprising nonviable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [group A, type 3] Su strain.

22. 22. The method of any one of claims 1 to 21, wherein the composition comprising nonviable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes.

23. 23. The method of any one of claims 1 to 22, wherein the composition comprising nonviable cells of Streptococcus pyogenes further comprises maltose, magnesium sulfate, potassium dihydrogen phosphate, sodium chloride, methionine, and benzylpenicillin.

24. 24. The method of any one of claims 1 to 23, wherein the composition comprising nonviable cells of Streptococcus pyogenes is a freeze-dried composition, and optionally the freeze-dried composition is reconstituted prior to administration.

25. 25. The method of any one of claims 1 to 24, wherein the triple-negative breast cancer is metastatic.

26. 26. The method of any one of claims 1 to 25, wherein the triple-negative breast cancer is recurrent.

27. 27. The method of any one of claims 1 to 26, wherein the triple-negative breast cancer is completely or partially resistant to a PD-1 inhibitor, a PD-L1 inhibitor, or both.

28. A pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat triple-negative breast cancer comprising nonviable cells of Streptococcus pyogenes.

29. A medicament for use in combination with an immune checkpoint inhibitor to treat triple-negative breast cancer containing nonviable cells of Streptococcus pyogenes.

30. 1. A method for treating non-muscle-invasive bladder cancer in a subject, comprising administering to the subject: (i) a composition comprising nonviable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

31. 31. The method of claim 30, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered intravesically, intratumorally, intravenously, intramuscularly, subcutaneously, or intraperitoneally.

32. 32. The method of claim 30 or 31, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of about 10 KE or greater.

33. 33. The method of claim 32, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of at least 20 KE.

34. 33. The method of claim 32, wherein the nonviable cells of Streptococcus pyogenes are present in the composition in an amount of at least 40 KE.

35. 35. The method of any one of claims 30-34, wherein the nonviable cells of Streptococcus pyogenes are administered to the subject at a dose of about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1 KE, about 0.5 KE, about 1 KE, about 2.5 KE, about 5 KE, about 10 KE, about 15 KE, about 20 KE, about 30 KE, about 40 KE, about 50 KE, about 60 KE, about 70 KE, about 80 KE, about 90 KE, or about 100 KE.

36. 36. The method of any one of claims 30 to 35, wherein the non-viable cells of Streptococcus pyogenes comprise cells of the Su strain of Streptococcus pyogenes.

37. 37. The method of any one of claims 30 to 36, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a nucleic acid molecule, a gene editing system, or a small molecule.

38. 38. The method of any one of claims 30-37, wherein the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 / PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

39. 39. The method of claim 38, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-1 inhibitor, optionally wherein the PD-1 inhibitor is an antibody.

40. 40. The method of claim 39, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrelimab, cemiplimab, sasanlimab, tislelizumab, or dostarlimab.

41. 39. The method of claim 38, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-L1 inhibitor, optionally wherein the PD-L1 inhibitor is an antibody.

42. 42. The method of claim 41, wherein the PD-L1 antibody comprises atezolizumab, durvalumab, embafolimab, or avelumab.

43. 39. The method of claim 38, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-L2 inhibitor, optionally wherein the PD-L2 inhibitor is an antibody.

44. 39. The method of claim 38, wherein the inhibitor of the PD-1 / PD-L1 / PD-L2 axis comprises a PD-1 inhibitor and a PD-L1 inhibitor, optionally wherein the PD-1 inhibitor and / or the PD-L1 inhibitor is an antibody.

45. 45. The method of any one of claims 30 to 44, wherein the non-muscle invasive bladder cancer is completely or partially resistant to a PD-1 inhibitor and / or a PD-L1 inhibitor.

46. 39. The method of claim 38, wherein the immune checkpoint inhibitor comprises a CTLA-4 inhibitor, optionally wherein the CTLA-4 inhibitor is an antibody.

47. 47. The method of claim 46, wherein the CTLA-4 antibody comprises ipilimumab, tremelimumab, or tubonlarimab.

48. 48. The method of any one of claims 30 to 47, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered at least one day before the immune checkpoint inhibitor.

49. 49. The method of any one of claims 30 to 48, further comprising administering to the subject an additional anti-cancer agent.

50. 50. The method of any one of claims 30-49, wherein the composition comprising nonviable cells of Streptococcus pyogenes is administered prior to, concurrently with, or subsequent to the immune checkpoint inhibitor.

51. 51. The method of any one of claims 30 to 50, wherein the composition comprising nonviable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [group A, type 3] Su strain.

52. 52. The method of any one of claims 30 to 51, wherein the composition comprising nonviable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes.

53. 53. The method of any one of claims 30-52, wherein the composition comprising nonviable cells of Streptococcus pyogenes further comprises maltose, magnesium sulfate, potassium dihydrogen phosphate, sodium chloride, methionine, and benzylpenicillin.

54. 54. The method of any one of claims 30 to 53, wherein the composition comprising nonviable cells of Streptococcus pyogenes is a freeze-dried composition, and optionally the freeze-dried composition is reconstituted prior to administration.

55. 55. The method of any one of claims 30-54, wherein the subject has low-grade non-muscle-invasive bladder cancer.

56. 56. The method of any one of claims 30-55, wherein the subject has high-grade non-muscle-invasive bladder cancer.

57. 55. The method of any one of claims 30 to 54, wherein the non-muscle invasive bladder cancer is identified as low risk.

58. 55. The method of any one of claims 30-54, wherein the non-muscle invasive bladder cancer is identified as intermediate risk.

59. 55. The method of any one of claims 30 to 54, wherein the non-muscle invasive bladder cancer is identified as high risk.

60. 55. The method of any one of claims 30-54, wherein the non-muscle invasive bladder cancer is identified as high-grade Ta or T1.

61. The method of any one of claims 30 to 54, wherein the non-muscle invasive bladder cancer is identified as carcinoma in situ (CIS) with or without Ta and / or T1.

62. 62. The method of any one of claims 30 to 61, wherein the non-muscle invasive bladder cancer is recurrent.

63. 63. The method of any one of claims 30 to 62, wherein the subject has not previously undergone BCG treatment.

64. 63. The method of any one of claims 30 to 62, wherein the subject has undergone appropriate BCG treatment.

65. 63. The method of any one of claims 30 to 62, wherein the subject is refractory to BCG treatment.

66. 63. The method of any one of claims 30 to 62, wherein the non-muscle invasive bladder cancer is BCG failed, BCG refractory, or BCG relapsing.

67. A pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat non-muscle invasive bladder cancer containing non-viable cells of Streptococcus pyogenes.

68. A medicament for use in combination with an immune checkpoint inhibitor to treat non-muscle invasive bladder cancer containing non-viable cells of Streptococcus pyogenes.

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