Combination of streptococcus pyogenes non-living cells and immune checkpoint inhibitor for treatment of triple negative breast cancer and non-muscular invasive bladder cancer

Abstract

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

Description

Background Art

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

[0002] Bladder cancer is the tenth most common cancer worldwide, with an incidence of almost half a million cases annually. Non-muscle invasive bladder cancer (NMIBC), defined as cancer confined to the bladder mucosa and submucosa, accounts for 75% of bladder cancer cases. The most common histological subtype is urothelial carcinoma. NMIBC includes papillary tumors within the mucosa (stage Ta), tumors that invade the lamina propria (stage T1), and flat, high-grade lesions known as carcinoma in situ (CIS). NMIBC is primarily treated with local endoscopic / intravesical therapy and surveillance.

[0003] The risk of non-muscle-invasive bladder cancer progressing to muscle invasion, or recurrence, varies depending on the grade and depth of the tumor. For example, at one end of the disease spectrum, low-grade Ta bladder cancer recurs in nearly two-thirds of cases but rarely (only about 6%) progresses to more aggressive disease, whereas NMIBC with high-risk features (including high-grade T1) has been reported to have a recurrence rate of almost 50%. Furthermore, this cancer progresses to muscle invasion in 1 in 5 patients, typically within 2 years of diagnosis.

[0004] BCG is the standard immunotherapy for NMIBC. After BCG failure, current treatment options are limited to radical cystectomy, which is the standard of care. For patients who are not suitable for or unwilling to undergo cystectomy, intravesical valrubicin and systemic pembrolizumab are currently the only two FDA-approved treatments for recurrent CIS. Given the limited options for BCG-unresponsive disease, particularly in light of the recent global BCG shortage and the high morbidity associated with radical cystectomy, there is an unmet need for treatments for high-risk NMIBC. Summary of the Invention

[0005] 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 Streptococcus pyogenes ( Streptococcus pyogenes ) a composition of non-viable cells; and (ii) an immune checkpoint inhibitor.

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

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

[0008] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 / PD-L2 pathway, 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 non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [Group A, Type 3] Su strain.

[0012] In some embodiments, the composition comprising non-viable S. pyogenes cells comprises benzylpenicillin-treated S. 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 non-viable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

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

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

[0016] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 / PD-L2 pathway, 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 a PD-1 / PD-L1 / PD-L2 axis inhibitor.

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

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

[0020] In some embodiments, the composition comprising non-viable S. pyogenes cells comprises benzylpenicillin-treated S. pyogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Evaluation of EMT6 triple-negative breast cancer tumor growth (before randomization) in 54 mice participating in the study.

[0022] Figure 2 : In vivo efficacy results of an exemplary composition comprising non-viable cells of Streptococcus pyogenes ("Composition 002" or "Comp. 002") in the EMT6 model.

[0023] Figures 3A to 3B :Intratumor( Figure 3A ) or intravenously ( Figure 3B ) In vivo efficacy results of delivery composition 002 as a single agent and in combination with anti-PD-1 antibody.

[0024] Figures 4A to 4B : Primary tumors of mice intratumorally administered Composition 002 as a single agent and in combination with anti-PD-1 antibody ( Figure 4A ) and spleen ( Figure 4B )'s weight measurements.

[0025] Figures 5A to 5B : Primary tumors in mice intravenously administered Composition 002 as a single agent and in combination with anti-PD-1 antibody ( Figure 5A ) and spleen ( Figure 5B ) weighing measurement data.

[0026] Figures 6A to 6B : Average body weight of Balb / c mice bearing EMT6 tumors (before randomization) Figure 6A ) and percentage change in body weight ( Figure 6B ) measurement results.

[0027] 7A to 7B : Average body weight of Balb / c mice bearing EMT6 tumors (after randomization, all groups) Figure 7A ) and percentage change in body weight ( Figure 7B ) measurement results.

[0028] Figures 8A to 8B : Intratumoral delivery group ( Figure 8A ) and intravenous delivery group ( Figure 8B ) were measured as percent body weight change (after randomization) in Balb / c mice bearing EMT6 tumors.

[0029] Figures 9A to 9B : Intravenous treatment group ( Figure 9A ) and intratumoral treatment group ( Figure 9B ) in the spleen (left) and tumor-TIL (right) + T cell % (gated on CD45 + CD3 + ) analysis.

[0030] FIG. 10A to FIG. 10B : Intravenous treatment group ( Figure 10A ) and intratumoral treatment group ( Figure 10B ) in the spleen (left) and tumor-TIL (right) + T cells (gated on CD45 + CD3 + CD8 - CD4 + ) analysis.

[0031] Figures 11A to 11B : Intravenous treatment group ( Figure 11A ) and intratumoral treatment group ( Figure 11B ) in the spleen (left) and tumor-TIL (right) + T cells (gated on CD45 + CD3 + CD4 CD8 + ) analysis.

[0032] FIG. 12A to FIG. 12B : Intravenous treatment group ( Figure 12A ) and intratumoral treatment group ( Figure 12B NK cells in the spleen (left) and tumor-TIL (right) of + Cells (gated on CD45 + CD3 - CD49b + -CD335 + ) analysis.

[0033] 13A to 13B : Intravenous treatment group ( Figure 13A ) and intratumoral treatment group ( Figure 13B Granulocytic MDSCs (gated on CD45) in spleen (left) and tumor-TIL (right) of + CD3 - CD11b +- Ly6G + Ly6C 低 ) and the percentage of monocytic MDSCs.

[0034] FIG. 14A to FIG. 14B : Intravenous treatment group ( Figure 14A ) and intratumoral treatment group ( Figure 14B ) of spleen (left) and tumor-TIL (right) neutrophil-MDSC cells (gated on CD45 + CD3 - CD11b +- Ly6G + Ly6C 低 ) Analysis of the ratio of MDSCs to monocytes.

[0035] FIG. 15A to FIG. 15B : Intravenous treatment group ( Figure 15A ) and intratumoral treatment group ( Figure 15B Regulatory T cells (gated on CD45) in spleen (left) and tumor-TIL (right) of + CD3 + CD4 + CD25 + Fox3 + ) analysis.

[0036] 16A to 16B : Intravenous treatment group ( Figure 16A ) and intratumoral treatment group ( Figure 16B TAMs (M1 / M2 ratio: gated on CD45) in spleen (left) and tumor-TIL (right) of + CD3 - f / 4 / 80 + CD206 - (M1) or CD45 + CD3 - f / 4 / 80+ CD206 + (M2)) analysis.

[0037] 17A to 17B : Intravenous treatment group ( Figure 17A ) and intratumoral treatment group ( Figure 17B PD-1 in spleen (left) and tumor-TIL (right) of + / 高 T cells (gated on CD45 + CD3 + ) analysis.

[0038] 18A to 18B : Intravenous treatment group ( Figure 18A ) and intratumoral treatment group ( Figure 18B PD-1 in spleen (left) and tumor-TIL (right) of + / 高 Macrophages (gated on CD45 + CD3 - f / 4 / 80 + ) analysis.

[0039] Figures 19A to 19B : Intravenous treatment group ( Figure 19A ) and intratumoral treatment group ( Figure 19B PD-1 in spleen (left) and tumor-TIL (right) of + / 高 T cells (gated on CD45 + CD3 + ) analysis.

[0040] FIG. 20A to FIG. 20B : Intravenous treatment group ( Figure 20A ) and intratumoral treatment group ( Figure 20B PD-1 in spleen (left) and tumor-TIL (right) of + / 高 Macrophages (gated on CD45 + CD3 - f / 4 / 80 + ) analysis.

[0041] Figures 21A to 21K : Summary of flow cytometric analysis data of composition 002 (dose 20 mg / Kg) in splenocytes and TILs of 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 ), Treg cells ( Figure 21F), macrophage M1 / M2 ratio ( Figure 21G ), PD-1 + T cells ( Figure 21H ), PD-L1 + T cells ( Figure 21I ), PD-L1 + Macrophages ( Figure 21J ) and PD-L1 + Macrophages ( Figure 21K ).

[0042] FIG. 22A to FIG. 22B : Antitumor efficacy of Composition 002 and anti-mPD-1 as single and combined therapies in the MBT-2 tumor model. Figure 22A ) Mean absolute tumor volume ± SEM over time; ( Figure 22B ) Individual absolute tumor volumes on day 7 (the last day of the study for all groups).

[0043] Figures 23A to 23F : Effect of Composition 002 and anti-mPD-1 treatment on leukocyte populations in subcutaneously (sc) implanted MBT-2 tumors in C3H mice - cell percentages. FC analysis was performed on cells isolated from MBT-2 tumors of 10 mice per group at the endpoint time point of day 10 (Groups 1 and 8) or day 8 (Groups 2 to 7). Cells were stained with antibody panels A and B as described in Example 2. The X-axis indicates the target population, presented as the percentage of each individual animal in the total population, which is shown in red text below the X-axis label. The horizontal bar in each data set indicates the group mean. ( Figure 23A ) Antibody Group A: CD45 cell;( Figure 23B ) Antibody Group A: CD4 + / CD8 + T cells and Treg cells;( Figure 23C ) Antibody group A: granulocytes and monocytes MDSC; ( Figure 23D ) Antibody Group B: CD45 CD3 CD11b cell;( Figure 23E ) Antibody group B: NK cells; ( Figure 23F ) Antibody group B: M1 and M2 macrophages.

[0044] Figures 24A to 24F: Effect of Composition 002 and anti-mPD-1 treatment on leukocyte populations of subcutaneously (sc) implanted MBT-2 tumors in C3H mice - cell counts. FC analysis was performed on cells isolated from MBT-2 tumors of 10 mice per group at the endpoint time point of day 10 (Groups 1 and 8) or day 8 (Groups 2 to 7). Cells were stained with antibody panels A and B as described in Example 2. The X-axis indicates the target population, presented as the cell count within the population for each individual animal, which is shown in red text below the X-axis label. The horizontal bar in each data set represents the group mean. ( Figure 24A ) Antibody Group A: CD45 cell;( Figure 24B ) Antibody Group A: CD4 + / CD8 + T cells and Treg cells;( Figure 24C ) Antibody group A: granulocytes and monocytes MDSC; ( Figure 24D ) Antibody Group B: CD45 CD3 CD11b cell;( Figure 24E ) Antibody group B: NK cells; ( Figure 24F ) Antibody group B: M1 and M2 macrophages.

[0045] Figure 25 : Effect of treatment on mouse body weight. Shown are the group mean relative body weights of all groups over time.

[0046] Figure 26 : Combination 002 treatment did not change CD4 + and CD8 + The number of T cells.

[0047] FIG. 27A to FIG. 27B :Composition 002 treated ( Figure 27A )CD4 + T cells and ( Figure 27B ) CD8 + Immune checkpoint molecule expression in T cells (mean of two donors). Data are presented as mean (two donors / three replicates) ± SD. = P < 0.01. = P < 0.001.

[0048] 28A to 28D Treatment with Composition 002 resulted in tumor cell apoptosis and the release of damage-associated molecular pattern molecules (DAMPs). MB49 cells were treated with various concentrations of Composition 002 for 24 hours. Mitoxantrone (1 μM) 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 expressed as mean ± SEM. Figure 28B : Calreticulin-positive MB49 cells quantified by flow cytometry. Figure 28C : Extracellular ATP (eATP) luminescence was measured and quantified using the following formula: [(triplicate cells 处理 RLU average - cell average - blank 培养基+药物 RLU) / (cell 未处理 RLU mean - cells - blank 培养基 RLU) × 100 - 100], where RLU represents background-subtracted luminescence. Figure 28D : After 24 h of treatment, supernatants were collected and HMGB1 was quantified using Lumit immunoassay. Figures 28B to 28D Mean fold change ± SEM of untreated controls are shown. One-way ANOVA with Tucky post hoc test , P<0.05; , P<0.01; , P<0.0001. N=3.

[0049] 29A to 29D MB49 cells previously exposed to Composition 002 resulted in dendritic cell (DC) maturation and higher phagocytosis rates. MB49 cells were pre-treated with different concentrations of Composition 002 for 24 hours. The drug was removed, and tumor cells were co-cultured with DC isolated from the bone marrow of C57bl / 6 mice for another 24 hours. CD86 positivity (a marker of cell maturation) was quantified by flow cytometry. Figure 29A ), CD80 positive ( Figure 29B ) and HLA-DR positive ( Figure 29C ) dendritic cells (CD11+). Figure 29D To assess phagocytosis, colocalization of pre-labeled tumor cell signals (DiO+) with DCs (CD11+) was quantified by flow cytometry. Dinaciclib (1 uM) was used as a positive control. Data are presented as mean ± SEM. One-way ANOVA with Tuckey post hoc test was performed. , P<0.05; , P<0.01; , P<0.0001. N=3.

[0050] FIG. 30A to FIG. 30B : Composition 002 effectively promotes immune-mediated tumor cell killing. Untreated cells (left column); cells treated with composition 002 (right column). Figure 30A: Human bladder cancer cells 5637 and RT112 were treated with composition 002 (0.2 KE / mL) for 72 hours. Figure 30B Human bladder cancer cells 5637 and RT112 were cocultured with PBMCs for 72 hours in the presence or absence of composition 002 (0.2 KE / mL). Tumor cell viability was assessed by flow cytometry using a live / dead dye. Data are presented as mean ± SEM. T-test. ; P<0.01; , P<0.0001. N=3; PBMCs from the same donor were used.

[0051] Figures 31A to 31B Treatment with Composition 002 induces Th1 and reduces Th2 cytokine release. Human bladder cancer cells 5637 and RT112 were co-cultured with PBMCs and Composition 002 (0.2 KE / mL) for 72 hours. Supernatants were collected, and a panel of proinflammatory cytokines was measured by electrochemiluminescence detection (MSD assay). Untreated cells (left column); cells treated with Composition 002 (right column). Figure 31A : Th1 cytokines (from left to right: IFN-γ, TNF-α and IL-12p70) and Figure 31B : Quantification of Th2 cytokines (from left to right: IL-13, IL-10, and IL-4). Data are expressed as mean ± SEM. T-test; ; P<0.01; , P<0.0001. N=3; PBMCs from the same donor were used.

[0052] Figures 32A to 32M Composition 002 induces T cell proliferation and activation, but also increases markers of exhaustion phenotype. T cells isolated from PBMC were treated with Composition 002 (0.2 KE / mL) for 72 hours. CD4+ T cell proliferation marker Ki67 ( Figure 32A ), and LAG3 ( Figure 32B )、CTLA4 ( Figure 32C )、PD-1 ( Figure 32D )、TIGIT ( Figure 32E )、Tim3 ( Figure 32F ) and FOXP3 ( Figure 32G ) marker. Figure 32H : CD8 T cell proliferation was assessed by quantification of Ki67-positive cells by flow cytometry. CTLA4 ( Figure 32I )、LAG3 ( Figure 32J )、PD-1( Figure 32K )、TIGIT ( Figure 32L ) and Tim3 ( Figure 32M ) markers. Data are presented as mean ± SEM of triplicates from two different donors. N = 6. T-test; ; P<0.01; , P<0.001.

[0053] Figures 33A to 33B Composition 002 activates T cells by increasing the release of IFN-γ and granzyme B. T cells isolated from PBMC were treated with Composition 002 (0.2 KE / mL) for 72 hours. IFN-γ ( Figure 33A ) and granzyme B ( Figure 33B Data are presented as mean ± SEM of triplicate samples from two different donors. N = 6. T-test; ; P<0.01; , P<0.001.

[0054] Figure 34 Treatment with Composition 002 induces PD-L1 expression in co-cultured 5637 bladder tumor cells. T cells isolated from PBMCs were co-cultured with human bladder cancer cells 5637 and Composition 002 (0.2 and 0.8 KE / mL) for 72 hours. PD-L1 signaling was quantified in pre-labeled 5637 cells by flow cytometry. Data are presented as mean ± SEM. N = 3. T-test. , P<0.05; ; P<0.01; , P<0.001.

[0055] Figures 35A to 35F :Anti-PD-L1 and anti-CTLA4 act synergistically with Composition 002. In vitro cytotoxicity was measured using an xCELLigence real-time cell analyzer. Human bladder cancer 5637 cells were seeded in 96-well E plates and grown for 78 hours. Subsequently, effector cells (human PBMC; effector / target ratio 6.6:1) were added and treated. Composition 002 was treated alone (0.8 KE / mL) or in combination with the following antibodies for approximately 65 hours in a co-culture environment (PBMC+tumor cells): ( Figures 35A to 35B ) Anti-PD-L1, ( Figures 35C to 35D ) Anti-CTLA-4, ( Figures 35E to 35F ) anti-PD-1. Irrelevant IgG4 and IgG1 were used as isotype controls. Impedance measurements were collected every 15 minutes using xCelligence RTCA eSight (Agilent). Data and Figure 35A 、 Figure 35C and Figure 35ESame as above, but each group was compared at a given time point (143 h:49 m:32 s) using histograms with error bars (respectively Figure 35B 、 Figure 35D and Figure 35F ) are shown. Data are expressed as mean ± SEM. N = 4. Statistical significance was determined using two-way analysis of variance (ANOVA) and Bonferroni post hoc test. , P<0.05; , P<0.01; P < 0.001; , P<.0001 (n=4).

[0056] Figures 36A to 36B Composition 002 treatment reduced tumor growth and prolonged animal survival in the anti-PD-1 sensitive MB49 subcutaneous mouse model. MB49 bladder cancer cells were subcutaneously implanted into C57bl / 6 mice. After 7 days, the animals were randomized and treated with Composition 002 intravenously (2, 0.4, or 0.8 KE / mouse) once weekly for 4 weeks. Figure 36A Tumor volume was analyzed every two weeks using a caliper. Data are presented as mean tumor volume. N = 10, two-way analysis of variance (ANOVA) with Bonferroni post hoc test; compared with untreated control (saline vehicle), ; P<0.01. Figure 36B : Kaplan Meier curve comparing the survival percentage between animals treated with Composition 002 and vehicle controls. N=10.

[0057] Figures 37A to 37E Composition 002 combined with the checkpoint inhibitor anti-PD-1 resulted in reduced tumor size in the MB49 subcutaneous mouse model. MB49 cells were subcutaneously implanted into C57bl / 6 mice. Six days later, the animals were randomized and treated with Composition 002 (0.4 KE / mouse) intravenously once weekly for four weeks and anti-PD-1 (10 mg / kg) intraperitoneally twice weekly for two weeks. Figure 37A Tumor volume was analyzed every two weeks using a caliper. Data are presented as mean ± SEM. N = 10, two-way analysis of variance (ANOVA) with Bonferroni post hoc test; compared with untreated controls (isotype controls), ; P<0.01; , P<0.0001. Figure 37BDetailed tumor volume curves comparing anti-PD-1 as monotherapy and combination therapy with Composition 002 + anti-PD-1. Data are presented as mean ± SEM. N = 10. Figure 37C : Bar graph analysis of mean tumor volumes of mice treated with anti-PD-1 as monotherapy or Composition 002 + anti-PD-1 combination on day 20 of treatment. Figure 37D : Tumor growth progression in mice treated with anti-PD-1. The light green line represents the individual mice involved in the study; the entire dark green line represents the average tumor volume. Figure 37E Figure 3: Tumor growth progression in mice treated with anti-PD-1. The light purple line represents the individual mice involved in the study, and the entire dark purple line represents the average tumor volume.

[0058] Figures 38A to 38B Treatment with Composition 002 reduced tumor growth in a PD-1-resistant triple-negative breast cancer model associated with increased PD-1 expression in T cells. Anti-PD-1-resistant triple-negative breast cancer cells (EMT6) were orthotopically implanted into female Balb / c mice. After 8 days, the mice were randomized and treated with twice-weekly intravenous injections of Composition 002 (0.4, 1, and 2 KE / mouse) for 3 weeks. Figure 38A Tumor volume was measured using a caliper. Data are presented as mean ± SEM. N = 6, T-test ; P<0.05. Figure 38B On day 22 after treatment initiation, mice were euthanized, tumors were harvested, and tumor-infiltrating immune cells were stained and analyzed by flow cytometry. Tumor-infiltrating T cells were analyzed for the presence of the PD-1 marker (from left to right: control; Composition 002 (0.4 KE / mouse); Composition 002 (1 KE / mouse); Composition 002 (2 KE / mouse)). Data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) with Dunnett's post hoc test was used. ,P<0.01, , P<0.001. N=3.

[0059] Figures 39A to 39B Composition 002, in combination with anti-PD-1, was shown to reduce tumor volume and weight in an orthotopic mouse model of EMT6 tumors resistant to anti-PD-1 therapy. Female Balb / c mice were orthotopically implanted with anti-PD-1-resistant triple-negative breast cancer cells. Eight days later, the mice were randomly divided into groups and treated with Composition 002 (10 mg / kg) intravenously twice a week for 3 weeks and anti-PD-1 (100 μg / mouse) intraperitoneally twice a week for 2 weeks. Figure 39ATumor volume was measured every two weeks using a caliper. Data are expressed as mean ± SEM. N = 6, one-way analysis of variance (ANOVA) with Bonferroni post hoc test; compared with untreated controls (saline control), ; P<0.05; , P<0.001. Figure 39B Tumor weights were quantified at the end of the study (day 30 after tumor implantation). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) with Tuckey post hoc test was used. , P<0.001. N=6.

[0060] Figures 40A to 40D : Combination therapy of composition 002 and anti-PD-1 reshapes the tumor microenvironment of the EMT6 orthotopic mouse model. Anti-PD-1 resistant triple-negative breast cancer cells were orthotopically implanted into female Balb / c mice. After 8 days, the mice were randomly divided into groups and treated with composition 002 intravenously (10 mg / Kg) twice a week for 3 weeks, and anti-PD-1 treatment (200 μg / Kg) was intraperitoneally injected twice a week for 2 weeks. On the 30th day after tumor implantation, the mice were euthanized, and the primary tumors were collected, processed and stained for flow cytometric analysis of tumor-infiltrating immune cells. Tumor infiltration analysis of T cells and PD-1 positive T cells (Figure 40a); Macrophage subsets ( Figure 40B ); C. Regulatory T cells ( Figure 40C ); Natural killer (NK) cells ( Figure 40D ). Data shown from left to right: control, anti-PD-1, Composition 002, and Composition 002 + anti-PD-1. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) with Tukey's post hoc test. , P<0.05, , P<0.01, , P<0.001; , P<0.0001. N=3. DETAILED DESCRIPTION

[0061] Before describing the present disclosure in more detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein. Additional definitions are set forth throughout the present disclosure.

[0062] In this specification, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the recited range, and where appropriate, include fractions (such as tenths and hundredths of integers) or sub-ranges thereof.

[0063] As used herein, unless otherwise indicated, the term "about" means ±20% of the indicated range, value, or structure.

[0064] It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of the alternatives.

[0065] As used herein, the terms "including," "having," and "comprising" are used synonymously and these terms and variations thereof are intended to be non-limiting.

[0066] "Optional" or "optionally" means that the subsequently described element, component, event or circumstance may or may not occur, and that the description includes instances where the element, component, event or circumstance occurs and instances where it does not.

[0067] The term "antibody" refers to an intact antibody comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to an antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including 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 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 intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem two-chain scFvs and tandem three-chain scFvs). Unless otherwise indicated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact antibodies 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.

[0068] As used herein, the term "immune checkpoint molecule" refers to one or more proteins, molecules, compounds or complexes that provide inhibitory signals to help control or suppress an immune response. For example, immune checkpoint molecules include partially or completely blocking immune stimulation; reducing, blocking or delaying immune activation; or increasing, activating or upregulating those molecules of immunosuppression. As used herein, "controlling or suppressing an immune response" means 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 inhibition can promote or allow the continued existence of hyperproliferative diseases or conditions (e.g., cancer, chronic infection).

[0069] 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-cells (e.g., T-cells), and T-cell remodeling. reg Cell or any combination thereof. In certain embodiments, immune checkpoint molecules can interact with ligands, such as by regulating (such as suppressing) antigen-specific T cell responses to initiate immunosuppressive signals. For example, T cells can express immune checkpoint receptors (such as PD-1, LAG3) on their surfaces, and antigen-presenting cells can express immune checkpoint receptor ligands (such as PD-L1, MHC / HLA molecules) on their surfaces. In further embodiments, immune checkpoint molecules are metabolic enzymes that suppress immune responses by locally depleting amino acids necessary for the survival and function of lymphocytes (particularly T cells). In further embodiments, immune checkpoint molecules can be signaling molecules, such as immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35).

[0070] Additionally, immune checkpoint molecules (e.g., IL-10) may cause a decrease in the expression or levels of major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules, which in turn may reduce antigen presentation, thereby reducing, arresting, or detectably preventing T cell activation and the corresponding immune response.

[0071] "Immune checkpoint inhibitors" refer to any molecules that can directly or indirectly change, interfere with, reduce, downregulate, block, inhibit, eliminate, or degrade the expression, amount, or activity of immune checkpoint molecules. Exemplary immune checkpoint inhibitors include small molecules, nucleic acid molecules (including vaccines (such as mRNA vaccines) and inhibitory nucleic acids (such as antisense oligonucleotides, siRNA, shRNA, and miRNA)), peptides, proteins, antibodies, or antigen-binding fragments thereof, fusion proteins, ribozymes, or gene editing systems.

[0072] As used herein, "triple-negative breast cancer" refers to a type of breast cancer that lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2.

[0073] As used herein, "non-muscle invasive bladder cancer" or "NMIBC" refers to urothelial cancer that is limited to the bladder mucosa and submucosa and does not invade or extend beyond the muscularis propria.

[0074] As used herein, the terms "treat" or "treatment" describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and include the administration of a composition of the present disclosure to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treat" may also include the treatment of animal models.

[0075] As used herein, the terms "treat" or "treatment" describe the management and care of a patient for the purpose of combating a disease, condition, or disorder, and include the administration of a composition of the present disclosure to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treat" may also include the treatment of animal models.

[0076] Compositions comprising non-viable cells of Streptococcus pyogenes

[0077] The present disclosure provides a composition comprising non-viable Streptococcus pyogenes cells and an immune checkpoint inhibitor for use in combination with the immune checkpoint inhibitor in treating a subject having triple-negative breast cancer or non-muscle invasive bladder cancer.

[0078] Streptococci are Gram-positive, catalase-negative, coagulase-negative cocci that occur in pairs or chains. They are divided into three groups based on the type of hemolysis on blood agar: beta-hemolytic (complete lysis of red blood cells), hemolytic (green hemolysis), and gamma-hemolytic (no hemolysis). Beta-hemolytic streptococci are characterized by group A streptococci (Streptococcus pyogenes) and group B streptococci (Streptococcus agalactiae). Streptococcus pyogenes is a Gram-positive, non-spore-forming, coccal bacterium that usually exists in cell chains or pairs. Streptococcus pyogenes is subdivided into 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 the M type (emm sequence type) of Streptococcus pyogenes. To date, 124 different M types have been identified (Facklam et al., Clin. Infect. Dis. 34, 1962). M11, M28, M12, M3, M11, and M6 are 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).

[0079] It is contemplated that any Streptococcus pyogenes ( S.pyogenes ) strain. In some embodiments of the present disclosure, the Streptococcus pyogenes strain used is selected by 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 highly virulent 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 avirulent. In some embodiments, the strain is avirulent due to a genetic mutation in a virulent strain.

[0080] In some embodiments, the composition comprises Streptococcus pyogenes M protein type 3.

[0081] In some embodiments, the composition comprises Streptococcus pyogenes (Group A, Type 3) Su strain.

[0082] In some embodiments of the present claims disclosure, the strain of Streptococcus pyogenes used is selected from the strains that have been deposited in the American Type Culture Collection (ATCC). In some embodiments, the present disclosure provides a mixture comprising more than one Streptococcus pyogenes strain. In some embodiments, the mixture comprises the Su strain and at least one additional Streptococcus pyogenes strain. Exemplary Streptococcus pyogenes strains contemplated for use in the present disclosure, including the Su strain, are described in Table A. Additional information for each strain is described at the following website: 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 A. Exemplary Streptococcus pyogenes strains

[0083] In some embodiments, the composition comprising inactivated Streptococcus pyogenes is a pharmaceutical composition, and optionally comprises at least one pharmaceutically acceptable excipient, such as a stabilizer, a buffer, a filler, an antioxidant, an osmotic pressure regulator, an antimicrobial or its any combination. As known to those skilled in the art, in some cases, component can have more than one activity, function or effect. For example, without wishing to be bound by theory, in some embodiments, some components (e.g., sodium chloride) can serve as a filler and an osmotic pressure regulator simultaneously.

[0084] In some embodiments, the composition is a lyophilized composition.

[0085] In some embodiments, the stabilizer is selected from the group consisting of magnesium hydroxide, calcium hydroxide, calcium carbonate, magnesium oxide, magnesium carbonate, magnesium sulfate, and combinations thereof.

[0086] 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.

[0087] 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), 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). (w / w) to about 0.50% (w / w).

[0088] 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 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).

[0089] 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).

[0090] In some embodiments, the stabilizer is present in the lyophilized composition at 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).

[0091] 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).

[0092] 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).

[0093] 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).

[0094] 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).

[0095] 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).

[0096] In some embodiments, the stabilizer is present in the lyophilized composition at about 4.50% (w / w) to about 5.00% (w / w).

[0097] In some embodiments, the buffer is a phosphate. In some embodiments, the buffer is selected from potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, and combinations thereof.

[0098] In some embodiments, the buffer is potassium dihydrogen phosphate. In some embodiments, the buffer is sodium phosphate.

[0099] In some embodiments, the buffering agent is present in the lyophilized composition at 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). (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), about 1.00% (w / w) to about 7.00% (w / w), about 1.00% (w / w) to about 6.00% (w / w), about 1.00% (w / w) to about (w / w) to about 5.00% (w / w), about 1.00% (w / w) to about 4.00% (w / w), about 1.00% (w / w) to about 3.00% (w / w), or about 1.00% (w / w) to about 2.00% (w / w).

[0100] 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 5.00% (w / w) to about 21.00% (w / w), about 5.00% (w / w) to about 20.00% (w / w), about 5.00% (w / w) to about 19.00% (w / w), about 5.00% (w / w) to about 18.00% (w / w), about 5.00% (w / w) to about 17.00% (w / w), about 5.00% (w / w) to about 16.00% (w / w). % (w / w), about 5.00% (w / w) to about 15.00% (w / w), about 5.00% (w / w) to about 14.00% (w / w), about 5.00% (w / w) to about 13.00% (w / w), about 5.00% (w / w) to about 12.00% (w / w), about 5.00% (w / w) to about 11.00% (w / w), 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 from 5.00% (w / w) to about 6.00% (w / w).

[0101] In some embodiments, the buffering agent is present in the lyophilized composition at 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), about 10.00% (w / w) to about 17.00% (w / w), about 10.00% (w / w) to about 19.00% (w / w), about 10.00% (w / w) to about 21.00% (w / w), about 10.00% (w / w) to about 22.00% (w / w), about 10.00% (w / w) to about 22.00% (w / w), about 10.00% (w / w) to about 23.00% (w / w), about (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).

[0102] In some embodiments, the buffer 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 21.00% (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). (w / w) to about 16.00% (w / w).

[0103] In some embodiments, the buffer 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).

[0104] In some embodiments, the filler is selected from the group consisting of sodium chloride, mannitol, sucrose, lactose, dextran, trehalose, glycine, maltose, and combinations thereof.

[0105] In some embodiments, the filler is sodium chloride. In some embodiments, the filler is mannitol. In some embodiments, the filler is sucrose. In some embodiments, the filler is lactose. In some embodiments, the filler is dextran. In some embodiments, the filler is trehalose. In some embodiments, the filler is maltose. In some embodiments, the filler is glycine.

[0106] 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).

[0107] 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).

[0108] 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).

[0109] 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).

[0110] 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).

[0111] In some embodiments, the bulking agent is present in the lyophilized composition at about 0.80% (w / w) to about 1.40% (w / w).

[0112] In some embodiments, the osmotic pressure regulator is selected from mannitol, D-mannitol, trehalose, α,α-trehalose dehydrate, sucrose, dextrose, sodium chloride, and maltose. In some embodiments, the osmotic pressure regulator is mannitol. In some embodiments, the osmotic pressure regulator is D-mannitol. In some embodiments, the osmotic pressure regulator is trehalose. In some embodiments, the osmotic pressure regulator is α,α-trehalose dihydrate. In another embodiment, the osmotic pressure regulator is sucrose. In another embodiment, the osmotic pressure regulator is dextrose. In another embodiment, the osmotic pressure regulator is sodium chloride. In some embodiments, the osmotic pressure regulator is maltose.

[0113] In some embodiments, the osmotic pressure regulating 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).

[0114] In some embodiments, the osmotic pressure regulating 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).

[0115] In some embodiments, the osmotic pressure regulating 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).

[0116] In some embodiments, the osmotic pressure regulating 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).

[0117] In some embodiments, the osmotic pressure regulating 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).

[0118] In some embodiments, the osmotic pressure regulating 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).

[0119] In some embodiments, the osmotic pressure regulating 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).

[0120] In some embodiments, the osmotic pressure regulating 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).

[0121] In some embodiments, the osmotic pressure regulating 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).

[0122] In some embodiments, the osmotic pressure regulating 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).

[0123] In some embodiments, the osmotic pressure regulating agent is present in the lyophilized composition at about 0.80% (w / w) to about 1.40% (w / w).

[0124] In some embodiments, when the lyophilized composition is reconstituted in water, the osmotic pressure regulator is present in the lyophilized composition in a calculated amount to produce an osmotic pressure equivalent to about 0.25% saline (w / w) to about 3% saline (w / w). In some embodiments, when the lyophilized composition is reconstituted in water, the osmotic pressure regulator is present in the lyophilized composition in a calculated amount to produce an osmotic pressure equivalent to about 1% saline (w / w) to about 2% saline (w / w). In some embodiments, the osmotic pressure regulator is present in the lyophilized composition in a calculated amount to produce a hypertonic solution. In some embodiments, the hypertonic solution has an osmotic pressure equivalent to greater than 0.9% saline (w / w).

[0125] In some embodiments, the antioxidant is selected from the group consisting of methionine, cysteine, histidine, arginine, lysine, and combinations thereof.

[0126] 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.

[0127] 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), 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 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), 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 (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).

[0128] 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).

[0129] 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), about 5.00% (w / w) to about 7.50% (w / w).

[0130] 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).

[0131] In some embodiments, the antioxidant is present in the lyophilized composition at about 6.00% (w / w) to about 8.00% (w / w).

[0132] In some embodiments, the antimicrobial agent is a penicillin. In some embodiments, the antimicrobial agent is selected from penicillin G or a pharmaceutically acceptable salt thereof, penicillin V or a pharmaceutically acceptable salt thereof, and combinations thereof.

[0133] In some embodiments, the antimicrobial agent is selected from the group consisting of penicillin G potassium (benzylpenicillin), penicillin V potassium (penicillin VK), and combinations thereof.

[0134] In some embodiments, the antimicrobial agent is penicillin G potassium (benzylpenicillin). In some embodiments, the antimicrobial agent is penicillin V.

[0135] 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).

[0136] 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).

[0137] 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).

[0138] 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).

[0139] 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).

[0140] 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°C to 38°C for more than 10 minutes, preferably 10 to 45 minutes, and then further heated at 38°C to 50°C for 20 to 60 minutes. An exemplary method for preparing a composition of the present disclosure includes starting with a primary culture of Streptococcus pyogenes; harvesting Streptococcus pyogenes cells from the primary culture; treating the Streptococcus pyogenes cells with hydrogen peroxide; washing the Streptococcus pyogenes cells; resuspending the Streptococcus pyogenes cells in a suspension medium; treating the Streptococcus pyogenes cells with benzylpenicillin; heating the Streptococcus pyogenes cells; preparing a final suspension of Streptococcus pyogenes cells; and lyophilizing the Streptococcus pyogenes cells.

[0141] In some embodiments, the lyophilized composition comprising inactivated Streptococcus pyogenes comprises: inactivated Streptococcus pyogenes (Group A, Type 3) Su strain; maltose, magnesium sulfate, potassium dihydrogen phosphate, 0.9% sodium chloride, methionine, and benzylpenicillin.

[0142] Table 13A shows exemplary quantitative formulations of different suggested dosage strengths of exemplary compositions comprising non-activated Streptococcus pyogenes. These compositions are based on lyophilized products. Exemplary quantitative formulations of exemplary compositions suspended in 0.9% saline are provided in Table 13B.

[0143] In some embodiments, the composition comprising non-activated S. pyogenes comprises whole S. pyogenes cells.

[0144] 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.

[0145] In some embodiments, the composition does not contain detectable viable S. pyogenes after storage for an extended period of time (e.g., at least two weeks, at least three weeks, at least four weeks, at least six weeks, at least two months, at least four months, at least six months, at least twelve months, at least eighteen months, at least twenty-four months, or at least thirty-six months) at 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).

[0146] In some embodiments, the composition does not comprise detectable viable S. pyogenes after storage at 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.

[0147] As used herein, a composition of the present disclosure is considered to have "retained efficacy" if, after a specified time period, the measured efficacy of the composition (as measured by conventional methods known in the art) is within the predetermined range of efficacy of an appropriate reference standard. In some embodiments, efficacy is calculated using a cytokine release assay. In some embodiments, the range is 20% to 180% of a reference standard. In some embodiments, the range is 40% to 160% of a reference standard. In some embodiments, the range is 50% to 150% of a reference standard. In some embodiments, the range is 60% to 140% of a reference standard. In some embodiments, the range is 70% to 130% of a reference standard. In some embodiments, the range is 80% to 120% of a reference standard. In a preferred embodiment, the range is 60% to 140% of a reference standard.

[0148] In some embodiments, after storage of the composition as described herein, the composition retains effectiveness 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.

[0149] In some embodiments, the composition retains potency for greater 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 2°C to about 8°C as described herein.

[0150] In some embodiments, the composition retains its effectiveness 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 under conditions of about 23°C to about 27°C and about 55% to about 65% relative humidity as described herein.

[0151] In some embodiments, the potency of the non-viable S. pyogenes cells and / or the relative percentage of non-viable S. pyogenes cells in the composition is stable, i.e., the potency is stable 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, or about 37 months after storage of the composition as described herein. , about 38 months, about 40 months, about 42 months, about 44 months, about 46 months or about 48 months, the potency of the non-viable S. pyogenes cells and / or the relative percentage of non-viable S. pyogenes cells in the composition does not change or changes by no 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% after 48 months.

[0152] In some embodiments, the composition comprising non-activated Streptococcus pyogenes is a lyophilized composition or a lyophilized formulation. In some embodiments, the composition comprising non-activated Streptococcus pyogenes is a lyophilized powder.

[0153] In some embodiments, the composition comprising inactivated Streptococcus pyogenes is OK-432 (Picibanil TM OK-432 is a freeze-dried biological product produced by treating and heating the Su strain of Streptococcus pyogenes (group A type 3) with benzylpenicillin. OK-432 undergoes no further processing, such as isolation, extraction, or purification. The bacterial cells remain intact. However, when administered to humans, their ability to proliferate is lost, and streptococcal infection does not occur.

[0154] In some embodiments, the composition comprising non-activated S. pyogenes is derived from genetically distinct group A type 3 S. pyogenes from the same master cell bank as OK-432.

[0155] Methods for preparing compositions comprising non-activated S. pyogenes are described in, for example, 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.

[0156] In some embodiments, the lyophilized compositions described herein can be further used to prepare a liquid composition of Streptococcus pyogenes. In some embodiments, the liquid composition is a suspension.

[0157] In some embodiments, liquid composition comprises the mixture of freeze-dried composition as described herein and water.In some embodiments, liquid composition comprises the mixture of freeze-dried composition as described herein and the NaCl aqueous 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 vehicle for freeze-dried composition is 0.9% sodium chloride solution.

[0158] In some embodiments, the lyophilized composition is suspended in a liquid (eg, isotonic sodium chloride solution) to prepare a suspension having a concentration of about 0.005 mg / mL to about 0.01 mg / mL.

[0159] In certain embodiments, Klinische Einheit (KE) is used as a unit of measurement for the dose of a composition comprising non-viable cells of Streptococcus pyogenes. 1 KE corresponds to a composition comprising approximately 1 × 10 8 0.1 mg of freeze-dried Streptococcus cells.

[0160] In some embodiments, the non-viable cells of S. 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.

[0161] In some embodiments, the non-viable cells of S. 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.

[0162] In some embodiments, the non-viable cells of S. 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.

[0163] In some embodiments, the non-viable cells of S. 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.

[0164] In some embodiments, non-viable cells of S. pyogenes are present in the lyophilized composition in an amount of non-activated S. pyogenes 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.

[0165] In some embodiments, non-viable S. pyogenes cells 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.

[0166] In some embodiments, non-viable S. pyogenes cells 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 non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in an amount of 15 ± 1 KE, 15 ± 2 KE, 15 ± 3 KE, or 15 ± 4 KE, 20 ± 1 KE, 20 ± 2 KE, 20 ± 3 KE, or 20 ± 4 KE, 30 ± 1 KE, 30 ± 2 KE, 30 ± 3 KE, or 30 ± 4 KE, 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE, 50 ± 1 KE, 50 ± 2 KE, 50 ± 3 KE, or 50 ± 4 KE, 60 ± 1 KE, 60 ± 2 KE, 60 ± 3 KE, or 60 ± 4 KE, 70 ± 1 KE, 70 ± 2 KE, 70 ± 3 KE, or 70 ± 4 KE, 80 ± 1 KE, 80 ± 2 KE, 80 ± 3 KE, or 80 ± 4 KE, 1 KE, 80 ± 2 KE, 80 ± 3 KE or 80 ± 4 KE, 90 ± 1 KE, 90 ± 2 KE, 90 ± 3 KE or 90 ± 4 KE, 100 ± 1 KE, 100 ± 2 KE, 100 ± 3 KE or 100 ± 4 KE or 150 ± 1 KE, 150 ± 2 KE, 150 ± 3 KE, or 150 ± 4 KE.

[0167] In some embodiments, the non-viable 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 non-viable cells of S. pyogenes are present in the lyophilized composition in an amount of 15 ± 1 KE, 15 ± 2 KE, 15 ± 3 KE, or 15 ± 4 KE, 20 ± 1 KE, 20 ± 2 KE, 20 ± 3 KE, or 20 ± 4 KE, 30 ± 3 KE, or 30 ± 4 KE, 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE, 50 ± 1 KE, 50 ± 2 KE, 50 ± 3 KE, or 50 ± 4 KE, 60 ± 1 KE, 60 ± 2 KE, 60 ± 3 KE, or 60 ± 4 KE.

[0168] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is about 15 KE, about 20 KE, about 30 KE, or about 40 KE. In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 15 ± 1 KE, 15 ± 2 KE, 15 ± 3 KE, or 15 ± 4 KE, 20 ± 1 KE, 20 ± 2 KE, 20 ± 3 KE, or 20 ± 4 KE, 30 ± 3 KE, or 30 ± 4 KE, 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE.

[0169] In some embodiments, the amount of non-viable cells of S. pyogenes present in the lyophilized composition is 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), 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 8.0% (w / w), at least 8.0% (w / w), at least 8.5 ... In some embodiments, the amount of non-viable S. pyogenes cells present in the lyophilized composition is 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%, 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.

[0170] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 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 5.5% (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 9.1% (w / w), about 9.2% (w / w), about 10.3% (w / w), about 11.1% (w / w), about 12.1% (w / w), about 13.1% (w / w), about 14.1% (w / w), about 15.1% (w / w), about 16.1% (w / w), about 17.1% (w / w), about 18.1% (w / w), about 19.1% (w / w), about 20.1% (w / w), about 21.1% (w / w), about 22.1% (w / w), about 23.1% (w / w), about 24.1% (w / w), about 25.1% (w / w), about 26.1% (w / w), about 27.1% (w / w), In some embodiments, the amount of non-viable S. pyogenes cells present in the lyophilized composition is 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.

[0171] In some embodiments, the amount of non-viable cells of S. pyogenes present in the lyophilized composition is 15 ± 1 KE, 15 ± 2 KE, 15 ± 3 KE, or 15 ± 4 KE and is 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.

[0172] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 20 ± 1 KE, 20 ± 2 KE, 20 ± 3 KE, or 20 ± 4 KE and is 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 amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 20 ± 1 KE, 20 ± 2 KE, 20 ± 3 KE, or 20 ± 4 KE, and is 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 amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE, and is about 3.9% (w / w) of the total weight of the lyophilized composition.

[0173] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 30 ± 1 KE, 30 ± 2 KE, 30 ± 3 KE, or 30 ± 4 KE and is 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.

[0174] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE and is 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 amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE, and is 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 amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 40 ± 1 KE, 40 ± 2 KE, 40 ± 3 KE, or 40 ± 4 KE, and is about 3.9% (w / w) of the total weight of the lyophilized composition.

[0175] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 50 ± 1 KE, 50 ± 2 KE, 50 ± 3 KE, or 50 ± 4 KE and is 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.

[0176] In some embodiments, the amount of non-viable cells of Streptococcus pyogenes present in the lyophilized composition is 60 ± 1 KE, 60 ± 2 KE, 60 ± 3 KE, or 60 ± 4 KE and is 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), 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.

[0177] In some embodiments, when the amount of non-viable Streptococcus pyogenes cells in the lyophilized composition increases, the relative percentage of non-viable Streptococcus pyogenes cells increases. For example, when the amount of non-viable Streptococcus pyogenes cells present in the lyophilized composition is 40 KE, 30 KE, 20 KE, 15 KE, or 10 KE, the relative percentage of non-viable Streptococcus pyogenes cells in the lyophilized composition is higher.

[0178] In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, 30 KE, 20 KE, or 15 KE, the relative percentage of non-viable 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 compared to 10 KE. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, 30 KE, 20 KE, or 15 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is higher by 20% ± 3%, 20% ± 2%, or 20% ± 1%, 25% ± 3%, 25% ± 2%, or 25% ± 1%, 30% ± 3%, 30 ± 2%, or 30% ± 1%, 35% ± 3%, 35% ± 2%, or 35% ± 1%, 40% ± 3%, 40% ± 2%, or 40% ± 1%, 45% ± 3%, 45% ± 2%, or 45% ± 1%, 50% ± 3%, 50% ± 2%, or 50% ± 1%. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, 30 KE, 20 KE, or 15 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 1%, 25% ± 1%, 30% ± 1%, 35% ± 1%, 40% ± 1%, 45% ± 1%, or 50% ± 1% higher than at 10 KE.

[0179] In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 15 KE, the relative percentage of non-viable 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 compared to 10 KE. In some embodiments, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 3%, 20% ± 2%, or 20% ± 1%, 25% ± 3%, 25% ± 2%, or 25% ± 1%, 30% ± 3%, 30 ± 2%, or 30% ± 1%, 35% ± 3%, 35% ± 2%, or 35% ± 1%, 40% ± 3%, 40% ± 2%, or 40% ± 1%, 45% ± 3%, 45% ± 2%, or 45% ± 1%, 50% ± 3%, 50% ± 2%, or 50% ± 1%, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 15 KE, as compared to 10 KE. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 15 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 1%, 25% ± 1%, 30% ± 1%, 35% ± 1%, 40% ± 1%, 45% ± 1%, or 50% ± 1% higher than at 10 KE.

[0180] In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 20 KE, the relative percentage of non-viable 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 than at 10 KE. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 20 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is higher by 20% ± 3%, 20% ± 2%, or 20% ± 1%, 25% ± 3%, 25% ± 2%, or 25% ± 1%, 30% ± 3%, 30 ± 2%, or 30% ± 1%, 35% ± 3%, 35% ± 2%, or 35% ± 1%, 40% ± 3%, 40% ± 2%, or 40% ± 1%, 45% ± 3%, 45% ± 2%, or 45% ± 1%, 50% ± 3%, 50% ± 2%, or 50% ± 1%, In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 20 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 1%, 25% ± 1%, 30% ± 1%, 35% ± 1%, 40% ± 1%, 45% ± 1%, or 50% ± 1% higher than at 10 KE.

[0181] In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 30 KE, the relative percentage of non-viable 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 than at 10 KE. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 30 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is higher by 20% ± 3%, 20% ± 2%, or 20% ± 1%, 25% ± 3%, 25% ± 2%, or 25% ± 1%, 30% ± 3%, 30 ± 2%, or 30% ± 1%, 35% ± 3%, 35% ± 2%, or 35% ± 1%, 40% ± 3%, 40% ± 2%, or 40% ± 1%, 45% ± 3%, 45% ± 2%, or 45% ± 1%, 50% ± 3%, 50% ± 2%, or 50% ± 1%. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 30 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 1%, 25% ± 1%, 30% ± 1%, 35% ± 1%, 40% ± 1%, 45% ± 1%, or 50% ± 1% higher than at 10 KE.

[0182] In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, the relative percentage of non-viable 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 compared to 10 KE. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is higher by 20% ± 3%, 20% ± 2%, or 20% ± 1%, 25% ± 3%, 25% ± 2%, or 25% ± 1%, 30% ± 3%, 30 ± 2%, or 30% ± 1%, 35% ± 3%, 35% ± 2%, or 35% ± 1%, 40% ± 3%, 40% ± 2%, or 40% ± 1%, 45% ± 3%, 45% ± 2%, or 45% ± 1%, 50% ± 3%, 50% ± 2%, or 50% ± 1%. In some embodiments, when the amount of non-viable S. pyogenes cells present in the lyophilized composition is 40 KE, the relative percentage of non-viable S. pyogenes cells in the lyophilized composition is 20% ± 1%, 25% ± 1%, 30% ± 1%, 35% ± 1%, 40% ± 1%, 45% ± 1%, or 50% ± 1% higher than at 10 KE.

[0183] Immune checkpoint inhibitors

[0184] The methods of the present disclosure provide for the administration of a composition comprising non-viable cells of Streptococcus pyogenes and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor can target the PD-1 / 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, IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.

[0185] In certain 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, siRNA, shRNA or miRNA. In some embodiments, the gene editing system is a CRISPR system, a TALEN system or a ZFN system.

[0186] In certain embodiments, the immune checkpoint inhibitor targets the PD-1 / PD-L1 / PD-L2 axis. In certain embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0187] In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a PD-1 inhibitor, such as a PD-1 specific antibody or a binding fragment thereof, such as Lambrilizumab, 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 ), Penpulimab (formerly known as AK105), Dostarlimab (Jemperli TM ), Camrelizumab, Airuika TM , SHR-1210), Prolgolimab (formerly known as BCD 100), Pucotenlimab (HX008), Serplulimab (HLX10), Cadonilimab (Ketanil TM, anti-PD-1 × anti-CTLA4 bispecific), Zimberelimab (AB122), Geptanolimab (GB226), Nofazinlimab, Sasanlimab (PF-06801591), QL-1604, Finotonlimab (formerly SCT I10A), BAT-1306, Budigalimab, Ezabenlimab, Peresolimab, Pimivalimab, Rulonilimab (formerly F520), Spartalizumab, MK-3475A, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, IAP-0971, IBI-318 (anti-PD-1 × anti-PD-L1 bispecific antibody), Ivonescimab (anti-PD-1 × anti-VEGFA bispecific antibody), Tebotelimab (anti-PD-1 × anti-LAG3 bispecific antibody), AZD-2936 (anti-TIGIT × anti-PD-1 bispecific antibody), EMB-02 (anti-PD-1 × anti-LAG3 bispecific antibody), Lorigerlimab (anti-PD-1 × anti-CTLA4 bispecific antibody), Vudalimab (anti-PD-1 × anti-CTLA4 bispecific antibody), Volrustomig (anti-PD-1 × anti-CTLA4 bispecific antibody), Fidasimtamab (anti-PD-1 × anti-HER2 bispecific antibody), Izuralimab (anti-PD-1 × anti-ICOS bispecific antibody), RG-6139 (anti-PD-1 × anti-LAG3 bispecific antibody) or any combination thereof.

[0188] In certain embodiments, a composition comprising non-viable Streptococcus pyogenes cells is used in combination with a PD-L1 specific antibody or a binding fragment thereof, such as BMS-936559, Durvalumab, Imfinzi, or the like. TM , MEDI4736), Atezolizumab (Tecentriq TM ,RG7446), Avelumab (Avelumab, Bavencio TM , MSB0010718C), Envafolimab (Enweida TM, KN035), Sugemalimab (Cejemly TM ), Cosibelimab (CK-301), Socazolimab (STI A1014), Tagitanlimab (HBM9167 or KL A167), MPDL3280A, SHR-1316, APL-502 (TQB 2450 or CBT 502), Danburstotug, Betifisolimab, Lesabelimab, Pacmilimab, Sudubrilimab (HS-636), LP-002, bintrafusp alfa (anti-PD-L1 antibody / TGFβRII extracellular domain fusion protein), SHR-1701 (anti-PD-L1 antibody / TGFβRII extracellular domain fusion protein), IBI-318 (anti-PD-1 × anti-PD-L1 bispecific antibody), KN-046 (anti-PD-L1 × anti-CTLA4 bispecific antibody), 6MW-3211 (anti-CD47 × anti-PD-L1 bispecific antibody), BNT-311 (anti-PD-L1 × anti-4-1BB bispecific antibody), Emfizatamab (anti-CD3e, anti-CD-19 anti-PD-L1, anti-4-1BB trispecific antibody), HB-0036 (anti-PD-L1 × anti-TIGIT bispecific antibody), HLX-301 (anti-TIGIT × anti-PD-L1 bispecific), or any combination thereof.

[0189] In certain embodiments, a composition comprising non-viable Streptococcus pyogenes cells is used in combination with a PD-L1 inhibitor, such as a vaccine, such as IO102 / IO103 (IDO peptide + PD-L1 peptide vaccine) or mRNA-4359 (IDO peptide + PD-L1 mRNA vaccine).

[0190] In certain embodiments, the composition comprising non-viable Streptococcus pyogenes cells is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, Tebotelimab (anti-PD-1 × anti-LAG3 bispecific antibody), RG-6139 (anti-PD-1 × anti-LAG3 bispecific antibody), or any combination thereof.

[0191] In certain embodiments, a composition comprising non-viable Streptococcus pyogenes cells is used in combination with a CTLA4 inhibitor. In specific 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), cadonilimab (anti-PD-1 × anti-CTLA4 bispecific antibody), KN-046 (anti-PD-1 × anti-CTLA4 bispecific antibody), lorigerlimab (anti-PD-1 × anti-CTLA4 bispecific antibody), vudalimab (anti-PD-1 × anti-CTLA4 bispecific antibody), volrustomig (anti-PD-1 × anti-CTLA4 bispecific antibody), or any combination thereof.

[0192] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a B7-H3-specific antibody or binding fragment thereof (eg, Enoblituzumab (MGA271), 376.96, or both).

[0193] In certain embodiments, a composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a B7-H4-specific antibody or binding fragment thereof (e.g., scFv or fusion protein thereof), such as Dangaj et al., Cancer Res. 73 :4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO2016 / 40724 and WO2013 / 025779, each of which is incorporated herein in its entirety.

[0194] In some embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a CD244 inhibitor.

[0195] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO2010 / 084158, the entirety of which is incorporated herein by reference.

[0196] In further embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a TIM3 inhibitor.

[0197] In further embodiments, the composition comprising non-viable S. pyogenes cells is used in combination with a Gal9 inhibitor.

[0198] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with an inhibitor of adenosine signaling (eg, an adenosine decoy receptor).

[0199] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with an A2aR inhibitor.

[0200] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a KIR inhibitor (eg, Lirilumab (BMS-986015)).

[0201] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0202] In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an IDO inhibitor, such as L-1-methyltryptophan, Epacadostat (INCB024360; Liu et al., Blood 115 :3520-30, 2010), Ebselen (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)-tira-pazamine, IO102 / IO103 (IDO peptide + PD-L1 peptide vaccine), or any combination thereof.

[0203] In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an arginase inhibitor, such as N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-1-arginine (de-NOHA), L-NOHA, 2(S)-amino-6-borohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.

[0204] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a VISTA inhibitor, such as CA-170 (Curis, Lexington, MA).

[0205] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with a LAIR1 inhibitor.

[0206] In certain embodiments, a composition comprising non-viable S. pyogenes cells is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0207] Treatment

[0208] Triple-negative breast cancer

[0209] The present disclosure provides methods of treating triple-negative breast cancer in a subject, comprising administering to the subject (i) a composition comprising non-viable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0210] In certain 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 an adult, teenager, child or infant.

[0211] In some embodiments, triple-negative breast cancer can be localized, regional, or metastatic. In some embodiments, triple-negative breast cancer can be newly diagnosed or recurrent cancer.

[0212] Triple-negative breast cancer can be divided into six distinct subtypes: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem cell-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR).

[0213] In some embodiments, the triple-negative breast cancer exhibits complete or partial resistance to a PD-1 inhibitor or a PD-L1 inhibitor.

[0214] Biological specimen can be obtained from object, for measuring the existence and / or level of estrogen receptor, progesterone receptor and HER2, or triple negative state." biological specimen " used herein can be a biopsy specimen from object or biological origin, a blood sample (from which serum or blood plasma can be prepared), a body fluid (such as lung lavage fluid, ascites, mucosal lavage fluid, synovial fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation. It is also possible to obtain a biological specimen from object before accepting any compositions comprising the non-viable cells of Streptococcus pyogenes.

[0215] The pharmaceutical composition can be administered in a manner suitable for the disease or condition to be treated (or prevented) as determined by those skilled in the art of medicine. The appropriate dosage and appropriate duration and frequency of administration of the composition will be determined by factors such as the patient's health, the patient's size (i.e., 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. Typically, appropriate dosages and treatment regimens provide compositions sufficient to provide therapeutic and / or preventative benefits (e.g., as described herein, including improved clinical outcomes, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival periods, or reduction in symptom severity). For prophylactic use, the dosage should be sufficient to prevent, delay, or reduce the severity of a disease or condition-related illness. The preventive benefits of the immunogenic compositions administered according to the methods described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and by analyzing the data obtained using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily implemented by those skilled in the art.

[0216] The pharmaceutical compositions described herein can be present in unit dose or multi-dose containers, such as sealed ampoules or vials. Such containers can be frozen before use to maintain the stability of the formulation. Suitable dosing and treatment regimens are developed for use of the specific compositions described herein in various treatment regimens, including, for example, parenteral or intravenous administration or formulations.

[0217] In some embodiments, the composition comprising inactivated Streptococcus pyogenes can be administered based on milligram or KE of dry cell mass. Therefore, can be with reference to mg or KE. In certain embodiments, the composition dosage comprising inactivated Streptococcus pyogenes is from about 0.1 KE to about 200 KE, from about 1 KE to about 100 KE, from about 5 KE to about 50 KE, or from about 0.1 KE, 0.5 KE, 1 KE, 2.5 KE, 5 KE, 10 KE, 15 KE, 20 KE, 30 KE, 40 KE, 50 KE, 60 KE, 70 KE, 80 KE, 90 KE, 100 KE, 125 KE, 150 KE, 175 KE or 200 KE. In certain embodiments, the unit dose of the composition comprising non-activated S. pyogenes is from about 0.01 mg to about 20 mg, or about 0.01 mg, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0218] In some embodiments, the composition comprising non-activated S. pyogenes can be administered to a subject daily, twice weekly, weekly, biweekly, or monthly.

[0219] If the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable non-toxic parenteral acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol or a mixture of polyethylene glycol and water. The aqueous solution or suspension may further comprise one or more buffers, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used to prepare any dosage unit formulation should be pharmaceutically pure and the amount used is substantially non-toxic. In addition, the active compound can be incorporated into sustained-release formulations and preparations. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit can contain a predetermined amount of recombinant cells or active compounds, which are calculated to produce the desired therapeutic effect in combination with an appropriate pharmaceutical carrier.

[0220] Typically, suitable dosages and treatment regimens provide active molecules or cells in an amount sufficient to provide therapeutic or preventive benefit. Such reactions can be monitored by establishing improved clinical outcomes (e.g., more frequent complete or partial remissions, or longer disease-free survival) in treated subjects compared to untreated subjects. An increase in pre-existing immune responses to tumor proteins is generally associated with improved clinical outcomes. Such immune responses can typically be assessed using standard proliferation, cytotoxicity, or cytokine assays, which can be performed using samples obtained from subjects before and after treatment.

[0221] In some aspects, the lyophilized pharmaceutical formulation is reconstituted prior to administration, eg, to form a liquid formulation of the disclosure.

[0222] In some embodiments, the formulation is administered to a subject using conventional modes of delivery including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intracystic, intramuscular, subcutaneous, or intratumoral administration.

[0223] In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is applied to the subject before administering the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 days or longer before administering the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject simultaneously with the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered on the same day as the immune checkpoint inhibitor. In some embodiments, after administering the immune checkpoint inhibitor, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered on the same day as the immune checkpoint inhibitor.

[0224] In other embodiments, the methods of the present disclosure further comprise administering an additional therapy 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; radiotherapy treatment; a cytokine; RNA interference therapy, or any combination thereof.

[0225] Exemplary monoclonal antibodies for cancer treatment include, for example, Galluzzi et al ., Oncotarget 5 (24):12472-12508, 2014, the monoclonal antibodies described therein are incorporated herein by reference in their entirety.

[0226] In certain embodiments, the combined treatment method includes further administering radiation therapy or surgery to the subject. Radiation therapy includes X-ray therapy (e.g., gamma radiation) and radiopharmaceutical therapy. Surgery and techniques suitable for treating a given cancer or non-inflammatory solid tumor can be combined with the modified immune cells of the present disclosure for use on a subject.

[0227] In certain embodiments, the combined treatment method comprises further administering to the subject a chemotherapeutic agent, including but not limited to chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (such as intercalators) and DNA repair inhibitors. 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)); antiproliferative / antimitotic agents, including vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and vinorelbine; inorelbine), 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, nitrogen mustard, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel, taxotere, temozolomide, teniposide, triethylenethiophosphamide, and etoposide). VP16)); antibiotics, such as dactinomycin D, daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which systemically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (altmethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), triazines - dacarbazinine (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 other thrombin inhibitors); fibrinolytics (e.g., tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (brefeldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF)). EGF 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 (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, actinomycin D, teniposide, epirubicin, edoxaban Topotecan, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin toxin, Pseudomonas aeruginosa exotoxin, pertussis adenylate cyclase toxin or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0228] Cytokines can be used to manipulate the host immune response to achieve anticancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42 :539, 2015. Cytokines used to promote 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, alone or in any combination.

[0229] Another cancer treatment approach involves reducing the expression of oncogenes and other genes required for cancer cell growth, maintenance, proliferation, and immune evasion. RNA interference, and in particular the use of microRNA (miRNA) and small inhibitory RNA (siRNA), provides a method for knocking out cancer gene expression. See, e.g., Larsson et al. et al., Cancer Treat. Rev. 16 :128, 2017.

[0230] In any embodiment disclosed herein, any therapeutic agent can be administered to a subject once or more than once during the course of treatment and can be administered to a subject in any order (e.g., simultaneously, concurrently, 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 specific form of the active ingredient; and the method of administration.

[0231] An effective amount of a treatment or pharmaceutical composition refers to an amount sufficient to achieve the desired clinical outcome or beneficial treatment as described herein within the required dosage and time period. An effective amount can be delivered in one or more administrations. If administered to a subject known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactically effective amount" can be used to describe an effective amount administered to a subject susceptible to developing a disease or disease state or to a subject at risk of developing a disease or disease state (e.g., recurrence) as a preventive course of treatment.

[0232] Non-muscle invasive bladder cancer

[0233] The present disclosure provides methods of treating non-muscle invasive bladder cancer in a subject, comprising administering to the subject (i) a composition comprising non-viable cells of Streptococcus pyogenes; and (ii) an immune checkpoint inhibitor.

[0234] In certain 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 an adult, teenager, child or infant.

[0235] Bladder cancer can be classified according to the traditional American Joint Committee on Cancer (AJCC) TNM staging. In the absence of lymph nodes (N phase) or distant metastasis (M phase), the depth of tumor invasion (T phase) is the most important determination method, and can be based on whether the tumor invades or exceeds the muscularis propria (muscle-invasive bladder cancer, MIBC) or does not invade or exceed the basal layer of the body (non-muscle-invasive bladder cancer, NMIBC) and is divided into two categories. Table B lists the primary tumor (T) staging in bladder cancer according to the AJCC. Tumors can be further classified according to histological grade (low or high). The World Health Organization (WHO) / International Society of Urological Pathology (ISUP) 2004 non-muscle-invasive urothelial tumor classification is provided in Table C. Table D provides the World Health Organization (WHO) 2004 urothelial carcinoma grading system.

[0236] Table B: Primary tumor (T) stage in bladder cancer

[0237] Table C: 2004 World Health Organization / International Association of Urological Pathologists: Classification of non-muscle invasive urothelial tumors

[0238] Table D: WHO 2004 urothelial carcinoma grades

[0239] In certain embodiments, a subject with non-muscle invasive bladder cancer has a stage Ta tumor. In certain embodiments, a subject with non-muscle invasive bladder cancer has a stage T1 tumor. In certain embodiments, a subject with non-muscle invasive bladder cancer has a stage Tis (CIS) tumor. In certain embodiments, a CIS tumor may or may not have Ta and / or T1.

[0240] In certain embodiments, the subject has papillary urothelioma of low malignant potential (PUNLMP). In certain embodiments, the subject has low-grade non-muscle invasive bladder cancer. In certain embodiments, the subject has high-grade non-muscle invasive bladder cancer. In certain embodiments, the subject has high-grade Ta non-muscle invasive bladder cancer. In certain embodiments, the subject has high-grade T1 non-muscle invasive bladder cancer.

[0241] Non-muscle-invasive bladder cancer can also be divided into three different risk categories based on the American Urological Association (AUA) and / or European Association of Urology (EAU) guidelines. NMIBC risk stratification groups and criteria are provided in Table E.

[0242] Table E: According to the American and European Associations of Urology

[0243] Definition of risk stratification groups for non-muscle invasive bladder cancer

[0244] LG = low-grade; PUNLMP = papillary urothelioma of low malignant potential; C HG = high grade; CIS = carcinoma in situ; LVI = lymphovascular invasion

[0245] In certain embodiments, the subject has non-muscle invasive bladder cancer that is identified as low risk according to the AUA and / or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that is identified as intermediate risk according to the AUA and / or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that is identified as high risk according to the AUA and / or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that is identified as highest risk according to the EUA guidelines. In certain embodiments, the subject with non-muscle invasive bladder cancer has lymphovascular invasion.

[0246] Intravesical Bacillus Calmette-Guérin (BCG) is a standard therapy for high-risk non-muscle invasive bladder cancer in patients who have not received BCG treatment, such as BCG treatment after surgical resection or tumor ablation. In certain embodiments, the subject suffering from non-muscle invasive bladder cancer has not received prior BCG therapy (BCG naïve). In certain embodiments, the subject suffering from non-muscle invasive bladder cancer has received sufficient BCG treatment. In certain embodiments, the subject suffering from non-muscle invasive bladder cancer is unresponsive to BCG therapy. Definitions of BCG unresponsive disease and sufficient BCG treatment are provided in Table F.

[0247] Table F: Key definitions and adequate BCG treatment for BCG-unresponsive non-muscle invasive bladder cancer

[0248] Other terms used to describe clinical scenarios in which BCG is not successful in treating high-risk non-muscle invasive bladder cancer and is no longer a treatment option include BCG failure (wherein muscle invasive bladder cancer is detected), BCG refractory (high-risk lesions are detected during or after adequate treatment of 3 or 6 months of treatment), BCG recurrent (tumor is detected after an initial response after completion of treatment), and BCG treatment is inadequate (the patient does not receive a full BCG dose due to intolerance or unsuitability of the BCG drug). In certain embodiments, the subject with non-muscle invasive bladder cancer suffers from BCG failure NMIBC. In certain embodiments, the subject with non-muscle invasive bladder cancer suffers from BCG refractory NMIBC. In certain embodiments, the subject with non-muscle invasive bladder cancer suffers from BCG recurrent NMIBC. In certain embodiments, the subject with non-muscle invasive bladder cancer is inadequately treated with BCG.

[0249] In certain embodiments, the subject with non-muscle invasive bladder cancer has not undergone radical cystectomy. In certain embodiments, the subject with non-muscle invasive bladder cancer is not a candidate for radical cystectomy.

[0250] In some embodiments, the non-muscle invasive bladder cancer may be a newly diagnosed or recurrent cancer.

[0251] In some embodiments, the non-muscle invasive bladder cancer exhibits complete or partial resistance to a PD-1 inhibitor or a PD-L1 inhibitor.

[0252] A biological sample can be obtained from a subject for determining the presence and / or stage or risk level of non-muscle invasive bladder cancer. As used herein, a "biological sample" can be a biopsy sample, a blood sample (from which serum or plasma can be prepared), a body fluid (e.g., urine, mucosal washes), 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 obtained from a subject prior to receiving any composition comprising non-viable Streptococcus pyogenes cells.

[0253] The pharmaceutical composition can be administered in a manner suitable for the disease or condition to be treated (or prevented) as determined by those skilled in the art of medicine. The appropriate dosage and appropriate duration and frequency of administration of the composition will be determined by factors such as the patient's health, the patient's size (i.e., 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. Typically, appropriate dosages and treatment regimens provide compositions sufficient to provide therapeutic and / or preventative benefits (e.g., as described herein, including improved clinical outcomes, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival periods, or reduction in symptom severity). For prophylactic use, the dosage should be sufficient to prevent, delay, or reduce the severity of the disease associated with the disease or condition. The preventive benefits of the immunogenic compositions administered according to the methods described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and by analyzing the data obtained using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily implemented by those skilled in the art.

[0254] The pharmaceutical compositions described herein can be presented in unit dose or multi-dose containers, such as sealed ampoules or vials. Such containers can be refrigerated prior to use to maintain the stability of the formulation. Suitable dosing and treatment regimens are developed for use of the specific compositions described herein in various therapeutic regimens, including, for example, parenteral or intravenous administration or formulation.

[0255] In some embodiments, the composition comprising inactivated Streptococcus pyogenes can be administered based on milligram or KE of dry cell mass. Therefore, can be with reference to mg or KE. In certain embodiments, the dosage of the composition comprising inactivated Streptococcus pyogenes is from about 0.1 KE to about 200 KE, from about 1 KE to about 100 KE, from about 5 KE to about 50 KE, or from about 0.1 KE, 0.5 KE, 1 KE, 2.5 KE, 5 KE, 10 KE, 15 KE, 20 KE, 30 KE, 40 KE, 50 KE, 60 KE, 70 KE, 80 KE, 90 KE, 100 KE, 125 KE, 150 KE, 175 KE or 200 KE. In certain embodiments, the unit dose of the composition comprising non-activated S. pyogenes is from about 0.01 mg to about 20 mg, or about 0.01 mg, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg.

[0256] In some embodiments, the composition comprising non-activated S. pyogenes can be administered to a subject daily, twice weekly, weekly, biweekly, or monthly.

[0257] If the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable non-toxic parenteral acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol or a mixture of polyethylene glycol and water. The aqueous solution or suspension may further comprise one or more buffers, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used to prepare any dosage unit formulation should be pharmaceutically pure and the amount used is substantially non-toxic. In addition, the active compound can be incorporated into sustained-release formulations and preparations. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit can contain a predetermined amount of recombinant cells or active compounds, which are calculated to produce the desired therapeutic effect in combination with an appropriate pharmaceutical carrier.

[0258] Typically, suitable dosages and treatment regimens provide active molecules or cells in an amount sufficient to provide therapeutic or preventive benefit. Such responses can be monitored by establishing improved clinical outcomes (e.g., more frequent complete or partial remissions, or longer disease-free survival) in treated subjects compared to untreated subjects. An increase in pre-existing immune responses to tumor proteins is generally associated with improved clinical outcomes. Such immune responses can typically be assessed using standard proliferation, cytotoxicity, or cytokine assays, which can be performed using samples obtained from subjects before and after treatment.

[0259] In some aspects, the lyophilized pharmaceutical formulation is reconstituted prior to administration, eg, to form a liquid formulation of the disclosure.

[0260] In some embodiments, the formulation is administered to a subject using conventional modes of delivery including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intracystic, intramuscular, subcutaneous, or intratumoral administration.

[0261] In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is applied to the subject before administering the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 days or longer before administering the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject simultaneously with the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered on the same day as the immune checkpoint inhibitor. In some embodiments, after administering the immune checkpoint inhibitor, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject. For example, the composition comprising non-viable cells of Streptococcus pyogenes can be administered on the same day as the immune checkpoint inhibitor.

[0262] In other embodiments, the methods of the present disclosure further comprise administering an additional therapy 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; radiotherapy treatment; cytokines; RNA interference therapy; a cancer vaccine, or any combination thereof.

[0263] Exemplary monoclonal antibodies for cancer treatment include, for example, Galluzzi et al ., Oncotarget 5 (24):12472-12508, 2014, the monoclonal antibodies described therein are incorporated herein by reference in their entirety.

[0264] In certain embodiments, the combined treatment method includes further administering radiation therapy or surgery to the subject. Radiation therapy includes X-ray therapy (such as gamma-radiation) and radiopharmaceutical therapy. Surgery and techniques suitable for treating a given cancer or non-inflammatory solid tumor can be combined with the modified immune cells of the present disclosure for use on a subject.

[0265] In certain embodiments, the combination treatment method comprises further administering BCG therapy to the subject.

[0266] In certain embodiments, the combined treatment method comprises further administering to the subject a chemotherapeutic agent, including but not limited to chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (such as intercalators) and DNA repair inhibitors. 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); antiproliferative / antimitotic agents, including vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and vinorelbine; epipodophyllotoxins (etoposide); , teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide (cytoxan), daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolomide, teniposide, triethylenethiophosphoramide, and etoposide, VP16); antibiotics, such as actinomycin D, daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which systemically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize asparagine themselves); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (altmethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), and triazines (dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, and aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, and nilutamide), and aromatase inhibitors (letrozole, anastrozole; anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (brefeldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil, and sirolimus). mofetil); antiangiogenic compounds (TNP470, 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 (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, doxycycline); Erythromycin, actinomycin D, teniposide (eniposide), epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin toxin, Pseudomonas aeruginosa exotoxin, pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0267] Cytokines can be used to manipulate the host immune response to achieve anticancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42 :539, 2015. Cytokines used to promote 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, alone or in any combination.

[0268] Another cancer treatment approach involves reducing the expression of oncogenes and other genes required for cancer cell growth, maintenance, proliferation, and immune evasion. RNA interference, and in particular the use of microRNA (miRNA) and small inhibitory RNA (siRNA), provides a method for knocking down cancer gene expression. See, e.g., Larsson et al. et al., Cancer Treat. Rev. 16 :128,2017.

[0269] In any embodiment disclosed herein, any therapeutic agent can be administered to a subject once or more than once during the course of treatment and can be administered to a subject in any order (e.g., simultaneously, concurrently, 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 specific form of the active ingredient; and the method of administration.

[0270] An effective amount of a treatment or pharmaceutical composition refers to an amount sufficient to achieve the desired clinical outcome or beneficial treatment as described herein within the required dosage and time period. An effective amount can be delivered in one or more administrations. If administered to a subject known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactically effective amount" can be used to describe an effective amount administered to a subject susceptible to developing a disease or disease state or to a subject at risk of developing a disease or disease state (e.g., recurrence) as a preventive course of treatment.

[0271] Example 1: In vivo efficacy of non-viable Streptococcus pyogenes cells as monotherapy and in combination with anti-mPD-1 in a triple-negative EMT6 breast cancer model

[0272] Materials and methods

[0273] Animals. Sixty-five 7-week-old female Balb / c mice were purchased from Jackson Laboratory and housed in cages of 5 mice per cage upon arrival, with food and water provided ad libitum in an indoor animal facility. Fifty-four mice were randomized to participate in the study, and an additional 11 mice (injected with tumor cells) were used for randomization purposes only. After one week of acclimatization, all mice confirmed to be healthy were weighed on day 0, before tumor cell implantation, and every two weeks thereafter until the end of the study.

[0274] Treatment. A few days before the start of the study, Composition 002 ("Comp. 002" or "002") was received as a lyophilized material in individual vials in a refrigerated box. There were 49 vials, each containing 20 KE, equivalent to 2 mg of drug. Composition 002 is a lyophilized biological preparation for administration that contains non-activated Streptococcus pyogenes (group A, type 3) Su strain cells treated with hydrogen peroxide and benzylpenicillin according to the present disclosure. Composition 002 was manufactured using the same master cell bank as OK-432 (Picibanil®).

[0275] Store all vials in a refrigerator (at 4°C) at the recommended temperature of 2°C to 8°C. Freshly reconstitute each vial with 1 ml of 0.9% sterile saline solution before administration to mice on each treatment day. Discard any remaining dissolved volume in the vial.

[0276] Anti-PD-1 antibody (CD279, clone RMP1-14, Catalog No. BP0146) and corresponding IgG control (isotype rat IgG2a, kappa (clone 2A3, Catalog No. BP0089)) were purchased from BioXcell. Shipping was delayed by several days, resulting in administration to mice several days after the scheduled dosing time.

[0277] Tumor Cells and In Vitro Culture. Cryopreserved vials of EMT6 mouse triple-negative breast cancer cells were thawed and incubated in sterile DMEM supplemented with sterile 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Cells were passaged in culture at a constant split ratio of approximately 1:6, meaning that cells were always harvested at the same confluency (approximately 70% to 80%) at the exponential growth phase at each passage before being re-plated in new T75 flasks.

[0278] The conditions for culturing and collecting cells were kept standardized to minimize any variability. Cells were passaged for two days before the scheduled injection into mice. Each passage and on the day of implantation into mice, cells were briefly treated (1 to 2 minutes) with warm trypsin / EDTA solution, followed by addition of sterile culture medium containing 10% serum, and washed twice in sterile serum-free culture medium to harvest the cells.

[0279] Keep the cell suspension on ice during preparation until the time of injection and to maintain cell viability and adherence during injection. After the final centrifugation, count the cells and resuspend them appropriately in sterile serum-free medium to a final concentration of 2.0 × 10 6 / ml, and injected 2 × 10 5 The calculated cell viability was 99%, confirming that highly viable cells were engrafted in the mice.

[0280] Sample processing for flow cytometric (FACS) analysis of splenocytes and tumor leukemia cells (TILs): Spleens were collected from a subset of three mice in each group under sterile conditions and placed in sterile cold culture medium. Single-cell suspensions of splenocytes (SPLCs) were prepared by squeezing them with a 3 ml syringe plunger and passing them through a 40 μm cell strainer. SPLCs were then processed and stained for FACS analysis.

[0281] Tumors were also collected from the same subset of 3 mice in each group and prepared by mechanical dissociation followed by treatment with collagenase D for 30 minutes at a final concentration of 2.5 mg / ml, freshly prepared just before use, to prepare tumor-infiltrating lymphocytes (TILs). The cell suspension was filtered through a 70 μM cell strainer, washed in sterile HBSS containing 2% FBS, and stained for FACS analysis.

[0282] Antibody staining and flow cytometric analysis: Before processing and staining samples for FACS analysis, purchase antibody panels and other related reagents for flow cytometry from Biolegend, BD Bioscience, and ThermoFisher Scientifics.

[0283] Immunophenotyping for FACS analysis (i.e., including CD8 + T cells, regulatory CD4 + T cells, NK cells, MDSCs, Tregs, and macrophages) and selected staining panels are described in the table below.

[0284] All samples were analyzed by LSRFortessa TM The cells were analyzed by flow cytometry (BD Biosciences). FACS profiles were further analyzed using Flojo software (treeStar).

[0285] Staining series (immunophenotyping)

[0286] Preparation of Single Cells for Single-Cell RNA (scRNA) Sequencing. At the end of the study, three selected tumors were processed for scRNA sequencing: one tumor from the intravenous control group (Group 2, Mouse #4), a second tumor from the intravenous injection of Composition 002, 10 mg / Kg group (Group 3, Mouse #6), and a third tumor from the intratumoral administration of Composition 002, 10 mg / Kg group (Group 4, Mouse #6).

[0287] In an incubator at 37°C, 5% CO2 atmosphere, the tumor was treated with collagenase type IV (final concentration 1 mg / ml) and DNAse (final concentration 100 units / ml) for about 1 hour to mechanically and enzymatically dissociate it into single cells. The sample was then gently washed by centrifugation at 980 rpm in HBSS containing 2% FBS, then treated with ACK for 3 minutes on ice, washed again and treated with a dead cell removal kit, and then resuspended in DMEM containing 20% FBS and 10% DMSO for cryopreservation. Each sample had two vials (1 ml each): each containing 2.5 × 10 6 cells (bottle #1) and 5.0 × 10 6 All samples contained highly viable single cells (average > 98%).

[0288] The samples were kept frozen until scRNA sequencing analysis.

[0289] Experimental Design and Results

[0290] In vivo implantation of tumor cells and treatment of tumor-bearing mice. After one week of acclimatization, 2×10 5 EMT tumor cells. Compared with the 5 × 10 5 The number of cells injected in this study was reduced compared to the number in the previous study, with the goal of slowing the rapid formation and growth of the tumor and preventing early ulceration. This change is important because the tumor size was approximately 100 mm when treatment began. 3 When randomized, mice can be grouped and more accurate and less variable efficacy results can be obtained.

[0291] Mice were carefully monitored twice weekly, and tumor growth was assessed by measuring length (L) and width (W) twice weekly with calipers. Tumor size was calculated using the formula (L × W²) / 2). Almost all tumors were measurable with calipers 6 days after inoculation and reached approximately 100 mm within 8 days. 3The average size of the tumor was 97% at this time. Tumor growth data before randomization are expressed as mean ± SEM and are Figure 1 Represented in the form of a diagram.

[0292] Eight days after tumor cell injection, 54 mice with tumors of comparable size were randomly divided into 9 groups of 6 mice each to ensure that the tumor means and standard deviations were similar between the groups before treatment exposure. The remaining 11 mice whose tumors were too small (or could not be measured with a caliper) or too large did not participate in the experiment. After randomization, mice in Group 1 and Group 2 (control) received vehicle saline by intratumoral and intravenous injection, respectively. Mice in Group 6 were administered intraperitoneally (ip) with anti-PD-1 antibody (200ug / mouse, 100ul volume) as a single agent, and mice in the control groups 1 and 2 received intraperitoneal injections of the same dose and volume of IgG control. Mice in Groups 4 and 8 were administered intravenously with 10 mg / Kg (100 μl volume) of Composition 002 alone and in combination with anti-PD-1 antibody, respectively.

[0293] Mice in treatment groups 3, 5, 7 and 9 were administered with 10 mg / Kg and 20 mg / Kg doses of Composition 002 as a single agent (Groups 3 and 9) and in combination with anti-PD-1 antibody (Groups 7 and 9) via intratumoral (it) route.

[0294] The mice in Groups 5 and 9 were initially planned to be dosed intravenously (iv) with a 20 mg / kg dose of Composition 002. However, this high intravenous dose was not tolerated, resulting in one mouse developing convulsions and dying rapidly after treatment. The dose was then reduced to 15 mg / kg, however, this still caused some adverse reactions: decreased activity, rough fur, and weight loss, which resolved within 2 to 3 days in some mice and were more severe in others. In trials with several additional mice, it was confirmed that 20 mg / kg of Composition 002 could be tolerated when administered intratumorally, and it was then decided to deliver this dose exclusively intratumorally. Therefore, the mice in Groups 5 and 9 received an initial dose of 15 mg / kg intravenously and then continued to be dosed at 20 mg / kg via the intratumoral route.

[0295] The treatment schedule for all mice with Composition 002 was twice a week, i.e., Monday and Thursday, for 3 weeks. The schedule for anti-PD-1 administration was also twice a week, one day after treatment with Composition 002. The treatment groups are described in Table 1 below.

[0296] Table 1: Experimental group arrangement

[0297] The volume of drug delivered intratumorally was 50 μl for the first week and increased to 100 μl thereafter as tumor size increased. The volume of 100 μl remained constant for all other groups throughout the study. It was previously planned to use a multi-side hole needle (purchased from Cook Medical) to improve intratumoral delivery through better penetration and uniform diffusion of the drug. However, when this needle was used in trials with saline in two additional mice, it was found that the performance of the needle was very poor due to the size and thickness of the needle making it difficult to insert without damaging the skin even at the smallest size. After the start of treatment, tumor growth continued to be assessed by caliper measurements twice a week. Data are expressed as tumor volume mean + standard deviation (STDV) and standard error (SEM), and growth curves for all groups (mean + SEM) are shown in Figure 5. Figure 2 Represented in the form of a diagram.

[0298] The percent tumor growth inhibition (%TGI) among the treatment groups was calculated and the results are reported in Table 2.

[0299] Table 2: Percent tumor growth inhibition (%TGI) measurements in Balb / c mice after 3 weeks of treatment with intravenous (IV) and intratumoral (IT) delivery of Composition 002 (Days 15 and 21)

[0300] Tumor efficacy data (mean + SEM) obtained from intravenous and intratumoral administration of Composition 002 are also presented in Figure 3A and Figure 3B Represented as a separate graphic.

[0301] Similar to our earlier efficacy trial results (unpublished), the data in this study showed that when delivered intravenously, a 10 mg / Kg dose of Composition 002 was more effective in inhibiting EMT6 tumor growth than intratumoral delivery. In addition, in combination with an anti-PD-1 antibody, systemic delivery of 10 mg / Kg of Composition 002 showed better anti-tumor activity than intratumoral administration of 10 mg / Kg or 20 mg / Kg. In addition, intratumoral treatment with Composition 002 did not cause a dose response, which is consistent with earlier observations. Due to the adverse events mentioned above, the intravenous delivery route could not evaluate the higher dose of 20 mg / Kg.

[0302] Thirty days after tumor cell implantation, i.e., 21 days after the start of treatment, primary tumors and spleens were collected from 3 mice in each group, processed, and stained for FACS analysis. Interestingly, mouse #2 in group 8 (composition 002 intravenously injected + anti-PD-1) showed a measurement of 130 mm on day 15 after the start of treatment. 3The tumor gradually decreased in size and regressed to non-palpable at the end of the study. The spleen was collected from this mouse and analyzed by FACS.

[0303] A total of 3 tumors were harvested and dissociated into single cells for scRNA analysis. Interestingly, the tumor of mouse #2 in group 8 (composition 002 intravenous injection + anti-PD-1) showed regression and became unmeasurable with calipers at the end of the study. For this mouse, only the spleen was collected and analyzed by FACS. The 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 also fixed in formalin. Details of the euthanasia date and collection sample processing are summarized in Table 3.

[0304] Table 3: Study Termination - Summary

[0305] Except for Group 4 (Composition 002 10 mg / Kg, IV) mouse #2 which had a 1 mm to 2 mm pulmonary metastasis on the lung surface, all groups of mice had no macroscopic metastasis in their lungs, however, this needs to be confirmed by histology.

[0306] The fixed samples were paraffin-embedded and blocks were prepared by the histopathology laboratory.

[0307] Tumors in several mice grew larger and developed black, bloody scabs, an indicator of the onset of ulceration, leading to their euthanasia a few days before the end of the study to comply with IACUC regulations. As shown in the data, and compared with our previous studies (unpublished), the inoculation of a smaller number of tumor cells resulted in less aggressive tumor growth and a reduction in the number of large, ulcerated tumors. Both the tumors and spleens were weighed, and the weight data, expressed as mean ± SEM, are reported in the graphs. Figures 4A to 4B (intratumoral delivery group) and Figures 5A to 5B (intravenous delivery group). Interestingly, an inverse correlation was found between tumor and spleen size: mice that showed a better response 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 had the largest spleen enlargement, indicating a stronger systemic immune response.

[0308] Monitoring of Mouse Weight and Health. Throughout the study, mice were carefully monitored daily for any abnormal signs, including respiratory distress, weakness, lethargy, difficulty moving, and / or weight changes (e.g., weight loss), large tumors (i.e., with a size restriction of >2,000), tumor ulceration, metastatic burden, and death.

[0309] Weight and health information were recorded weekly and provided in an Excel file. Weight data were expressed as mean and percentage change; Figure 6A and Figure 6B as well as Figure 7A and Figure 7B In the , graphical representations of these data are reported separately for all groups before and after randomization. Figure 7A and Figure 7B Separate graphical representations of the data for the intratumoral and intravenous drug delivery groups are shown in .

[0310] No mouse showed significant weight changes, i.e., >15% or >20% weight loss, however, accurate measurements could not be made for mice with the largest tumors or enlarged spleens because the weight gain of the tumors or spleens partially compensated for the reduced weight. There were two deaths, one in mouse #2 from Group 2 control and the other in mouse #6 from Group 8 (Composition 002 10 mg / Kg IV in combination with anti-PD-1 antibody); although the death of the control mouse was likely due to a large tumor with black blood crusts / ulcers, the cause of death of the other mouse may be due to the effects of the treatment, a strong immune response, or a combination of both.

[0311] For products with a diameter of 2,000 mm or more 3 Mice with size-restricted tumors were euthanized within 24 to 48 hr to allow for planned harvest and analysis and in compliance with IACUC protocol requirements.

[0312] Flow cytometric analysis of splenocytes (SPLN) and tumor infiltrating lymphocytes (TIL). After harvesting and processing tumors (TIL) and spleens (SPLC), the samples were stained and analyzed by flow cytometry to determine the effect of composition 002 on different types of immune cells according to the immunophenotype and selected combination in the above-mentioned procedure section. The individual replicates and mean + standard deviation for each sample are reported in Tables 4A to 7C. The percentages of T cell subsets in spleen and tumor-derived TILs were analyzed by flow cytometry. The flow cytometric data from the spleen of mouse #2 of the combined Group 8 (Composition 002 10 mg / Kg intravenous + anti-PD-1) showing tumor regression were similar to the mean values of the group in all immunophenotypes.

[0313] Table 4A: FACS analysis data of spleen from intratumoral group

[0314] Table 4B: FACS analysis data of spleen from intratumoral group

[0315] Table 4C: FACS analysis data of spleen from intratumoral group

[0316] Table 5A: FACS analysis data of TILs from the intratumoral group (repeated measurements)

[0317] Table 5B: FACS analysis data of TILs from the intratumoral group (repeated measurements)

[0318] Table 5C: FACS analysis data of TILs from the intratumoral group (repeated measurements)

[0319] Table 6A: FACS analysis data of spleens from the intravenous group

[0320] Table 6B: FACS analysis data of spleens from the intravenous group

[0321] Table 6C: FACS analysis data of spleens from the intravenous group

[0322] Table 7A: FACS analysis data of TILs from the intravenous group (repeated measurements)

[0323] Table 7B: FACS analysis data of TILs from the intravenous group (repeated measurements)

[0324] Table 7C: FACS analysis data of TILs from the intravenous group (repeated measurements)

[0325] As shown in Figures 9 to 19, graphical representations of the immune cell percentage data are shown in scatter plots. These results compare the two delivery routes of Composition 002 administered at a dose of 10 mg / kg. Statistical analysis was performed using unpaired t-tests and one-way ANOVA using GraphPad Prism 9 to determine significant differences in the expression of selected markers between the groups.

[0326] Figures 9A to 9B The results presented are similar to our previous study data, showing minimal or no statistically significant changes in the percentage of CD3+ T cells in all groups in the spleen cells and tumor TILs of mice treated intravenously with Composition 002, as well as in the tumors of mice treated intratumorally with the drug. However, this time, there was a consistent and significant increase in T cells in the spleens of mice that received 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 high dose of 20 mg / kg, had no such effect.

[0327] FIG. 10A to FIG. 10B The results shown show the effects of Composition 002 on the spleen and tumors, which are relatively consistent with our previous observations: there was an effect on the spleen after delivery of the drug as a single agent or in combination with anti-PD-1, regardless of the route, and there was only a trend of approximately 40% to 50% reduction in CD4+ T cells in tumors when the drug was administered intravenously. In this study, the data showed that after intravenous administration of Composition 002, the suppression of CD4+ T cells in tumors was smaller, more effective, and statistically significant, which was sufficient to compensate for the increase in CD4+ T caused by anti-PD-1 in combination therapy.

[0328] like Figures 11A to 11B As shown, intravenous administration of Composition 002 reduced the percentage of CD8+ T cells (having a CD45+ CD3+ CD4-CD8+ phenotype) in the spleen and tumors, with the reduction in the spleen being statistically significant compared to the control and anti-PD-1 treatment. In the combination group with intravenous delivery of Composition 002, the effect of anti-PD-1 as a single agent on increasing CD8+ T cells was significantly attenuated by Composition 002, resulting in lower levels of immune cells with this phenotype; this low level was comparable to the level measured after treatment with Composition 002 as a single agent. In mouse tumors treated intratumorally with Composition 002, Composition 002 alone did not have such a significant inhibitory effect, but the combination of Composition 002 and anti-PD-1 had a statistically significant synergistic effect compared to each single agent and the control.

[0329] When the percentage of NK cells (gated on CD45+CD3-CD49b+CD335+) was examined in splenocytes or TILs in all intravenous groups, no significant changes were observed compared to the control, except that Combination 002 or anti-PD-1 as single agents was significantly reduced in the spleen and further reduced in the combination group, as shown in Figure 3. FIG. 12A to FIG. 12B In tumors, there was no difference between intravenously administered Composition 002 or PD-1 as single agents and controls, but the increase in NK cells in the combination group was statistically significant.

[0330] The percentage of NK cells in the spleen did not differ between the intratumoral groups, but the percentage of NK cells in the tumor was statistically significantly reduced by anti-PD-1 in both the single-agent and combination groups.

[0331] To assess the effect of treatment on the immunosuppressive microenvironment of EMT6 tumors, the percentages of granulocyte-MDSCs and monocytic-MDSCs alone and the ratio of granulocyte-MDSCs to monocytic-MDSCs were calculated based on the myeloid-derived suppressor cell subsets granulocytes and monocytes, respectively. The data are shown in 13A to 13B and FIG. 14A to FIG. 14B These results suggest that in tumors, this ratio was unaffected by Composition 002 and was enhanced by anti-PD-L1 antibodies, which correlates with the lack of efficacy of PD-1 inhibitors; however, in the combination group, this ratio was significantly reduced, likely due to some indirect effects of Composition 002 delivered intravenously or intratumorally. In contrast, these effects were less pronounced in the spleen. Specifically, the MDSC / monocyte ratio increased in mice administered Composition 002 alone by either route, but not in combination with anti-PD-1.

[0332] exist FIG. 15A to FIG. 15B The CD45 + CD3-CD4 + CD25 + Foxp3 + Analysis of the data for regulatory T cells (Treg) phenotype showed that Composition 002 did not significantly change in the spleen whether it was administered intravenously or intratumorally, but had a statistically significant increase in tumors after intravenous delivery and a statistically significant decrease after administration of anti-PD-1 antibodies. The decrease induced by anti-PD-1 was reversed by combination with Composition 002.

[0333] Next, tumor-associated macrophages (TAMs) were assessed in three tumor subsets from all groups, gating on CD45 + CD3 - F4 / F80 + CD206 -(M1) or CD206 + (M2). Data in 16A to 16B The figure depicts findings similar to those of our previous study. Composition 002 treatment resulted in a significant and sustained increase in M1 macrophages and a significant decrease in M2 macrophages in both tumors and spleens, leading to a high M1 / M2 ratio in all Composition 002-treated groups, both in the IV and IT groups. This ratio remained elevated in the combination group, unaltered by PD-1 inhibition, leading to more effective anti-tumor activity.

[0334] In T cells (gated on CD45 + CD3 + ) were analyzed by FACS analysis of the immune checkpoint PD-1. 17A to 17B Compared to the control, treatment with Composition 002 increased the expression level of PD-1 in T cells, both in splenocytes and TILs, after either intravenous or intratumoral treatment. However, the increase in PD-1 expression levels in the spleen was higher and more statistically significant compared to the tumor, and PD-1 expression was stronger in the intravenous delivery group. The percentage of PD-1 in the spleen or tumor of mice treated with anti-PD-1 remained significantly lower than that of those given Composition 002, but the decrease induced by the anti-PD-1 antibody was statistically significant only in the spleen compared to the control. PD-1 expression remained elevated in the combination group compared to the single-agent groups, likely due to the augmentation effect of Composition 002.

[0335] like 18A to 18B As shown, intravenous delivery of composition 002 resulted in PD-1 + Macrophages (gated on CD45 + 、CD3-F4 / 80 + ) percentages were increased to a greater extent in tumors than in spleens, whereas intratumoral delivery did not. + There was no effect on the percentage of macrophages, but PD-1 in tumors was significantly increased compared with the control baseline in the intravenous group. + If the effects of Composition 002 and anti-PD-1 antibody were inconsistent between groups, this could be because only 3 samples were selected between the two delivery groups to determine the expression of PD-1. + Some differences in baseline macrophage levels.

[0336] In the combination group of the intravenous group, PD-1 + The levels of macrophages remained low in the tumors and were statistically different from the values measured after treatment with Composition 002.

[0337] like Figures 19A to 19BAs shown, T cells (gated on CD45) of mice treated intravenously with Composition 002 were significantly downregulated compared to controls. + CD3 + ) was higher in tumors than in the spleen and was not affected by anti-PD-1 antibodies. Statistically significant differences were found between control and anti-PD-1 treated tumors in the intratumoral group.

[0338] In the group using combination 002 and anti-PD-1, PD-L1 + The percentage of T cells remained high due to the increasing effect of Composition 002, similar to the PD-1 expression results in T cells.

[0339] Finally, in FIG. 20A to FIG. 20B PD-L1 reported in + Macrophage data analysis confirmed that Composition 002 induced consistent increases in both spleens and tumors in both the intravenous and intratumoral groups, unaltered by anti-PD-1 antibodies. This increase was stronger and more statistically significant in the spleen than in the tumor, and was maintained with the combination of Composition 002 and anti-PD-1 antibodies.

[0340] All flow cytometry analysis data described above focused on the comparison of one dose (10 mg / Kg) of Composition 002 administered intravenously or intratumorally to mice as a single agent and in combination with anti-PD-1 antibody.

[0341] In this study, a higher dose of Composition 002 (20 mg / Kg) was also tested via the intratumoral route and the data are reported in Figures 21A to 21K In most cases, the higher dose of composition 002 had no greater effect on the analyzed immunophenotypes than the lower dose, except for a slight increase (about 1.5 times) in the M1 / M2 ratio in T cells induced in the spleen, which was also statistically significant. In addition, compared with the combination of composition 002 and anti-PD-1 at a dose of 10 mg / Kg, the combination of composition 002 and anti-PD-1 at a dose of 20 mg / Kg was more effective in increasing M1 macrophages. Similarly, compared with the combination of low-dose composition 002 and anti-PD-1, the combination of high-dose composition 002 and anti-PD-1 caused a slightly higher increase in the PD-1 percentage of T cells in the spleen (statistically significant compared to control group 1). No such effect was observed in tumors by intratumoral delivery of both doses of composition 002.

[0342] in conclusion

[0343] Findings from this study have demonstrated significant inhibition of EMT6 tumor growth by Composition 002 as a single agent, which recapitulates our previously unpublished findings and demonstrates the excellent anti-tumor activity of the combination of Composition 002 and anti-PD-1 antibodies.

[0344] Consistent with our previous observations, intravenous drug delivery in this study was more effective than intratumoral administration. Intratumoral administration of Composition 002 to mice at doses of 10 mg / kg and 20 mg / kg did not elicit a dose-response effect. The higher dose of 20 mg / kg could not be tested via the intravenous route, as this has been shown to be unsafe. Therefore, unless drug penetration and distribution through tumor tissue are improved, it remains unknown whether increasing tumor exposure to higher-drug treatment regimens will lead to increased efficacy.

[0345] The most significant and consistent changes induced by Composition 002 were in tumor-associated macrophages (TAMs), with an increase in the M1 cell type and a decrease in the M2 cell type, accompanied by a higher percentage of PD-1 + / PD-L1 + T cells and PD-1 + / PD-L1 + Macrophages. The increase in the M1 / M2 ratio, consistent with the paradigm that M1 represents antitumor activity while M2 drives tumor progression, explains one mechanism of antitumor activity.

[0346] As reported in the literature, the EMT6 breast tumor model has a weak response to anti-PD-1 antibodies and, in this study, was shown to be completely resistant to this immune checkpoint inhibitor. Interestingly, despite the lack of efficacy of anti-PD-1 as a single agent, the combination of Composition 002 and anti-PD-1 antibodies further reduced tumor growth compared to monotherapy.

[0347] The higher expression of PD-1 on T cells in the treatment group may be 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 to target MDSCs to overcome anti-PD-1 resistance.

[0348] Furthermore, targeting PD-L1 in addition to PD-1 in combination with Composition 002 may be a better therapy for EMT6 and ultimately for triple-negative breast cancer in the clinic.

[0349] Example 2: In vivo efficacy of non-viable Streptococcus pyogenes cells as monotherapy and in combination with anti-mPD-1 in a syngeneic bladder cancer MBT-2 model implanted subcutaneously in C3H / HeN mice

[0350] The anti-tumor efficacy of Composition 002 (a lyophilized formulation 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 subcutaneous (sc) implantation of immunocompetent C3H / HeN mice in the mouse bladder tumor model MBT-2. The efficacy experiment was started with 8 groups of 15 mice each, and each mouse was given a subcutaneous injection of 2 mg / kg, 1 mg / kg and 0.5 mg / kg of lyophilized Streptococcus pyogenes alone or in combination with 5 mg / kg of anti-mPD-1 injected intraperitoneally (ip) twice a week. One group received 5 mg / kg of anti-mPD-1 treatment and one group received a vehicle of lyophilized Streptococcus pyogenes as a reference control. At the beginning of the experiment, the tumor volume ranged from 50 mm 3 Up to 150 mm 3 When it reaches more than 1,500 mm in the first animal 3 The experiment was concluded when the tumor volume reached the termination standard of . The experiment was divided into two groups: Groups 2 to 7 ended on day 8, while Groups 1 and 8 ended on day 10.

[0351] The anti-tumor efficacy of all groups was evaluated using the vehicle control group as a reference. Tumor samples collected at the end were used for downstream analysis of tumor infiltrating leukocytes (TILs). CD4 T cells in the tumors of 10 animals in each group were evaluated by FC analysis using two predetermined marker panels. + and CD8 + T cell, Treg, granulocyte-derived cytokine-stimulating factor (MDSC), monocytic-derived cytokine (MDSC), NK cell, and M1 / M2 macrophage populations were analyzed using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A to assess changes in various cytokines in EDTA plasma and tumor samples collected during and at the end of the experiment. These data are reported separately.

[0352] Composition 002 at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg in monotherapy and anti-mPD-1 at 5 mg / kg in monotherapy showed no anti-tumor activity against the MBT-2 tumor model in this study. Composition 002 at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg in combination with anti-mPD-1 were also ineffective against the MBT-2 tumor model in this study, and no statistically significant differences in tumor volume were observed between any test group and the vehicle control group (Kruskal-Wallis combined with Dunn post hoc test).

[0353] FC analysis of cells isolated from MBT-2 tumors at the end time points showed that CD45 +The percentage of cells was lower than that of the control group. + and CD8 + The intragroup variation of cell percentage is very high. Compared with the control group, the Treg in the three monotherapy groups of composition 002 and anti-mPD-1 monotherapy group and 1 mg / kg composition 002 / anti-mPD-1 group increased significantly (Kruskal-Wallis combined with Dunn post hoc test). Compared with the control, except for 2 mg / kg composition 002 monotherapy and 0.5 mg / kg composition 002 / anti-mPD-1 group, the granulocyte MDSC percentage in all test groups was significantly lower, while the monocyte MDSC percentage was significantly higher. For NK cells, no significant differences were observed between the test group and the control group. Compared with the control group, except for 0.5 mg / kg composition 002 / anti-mPD-1 group, the M1 macrophage frequency in all test groups was significantly higher, while the M2 macrophage frequency was significantly lower.

[0354] After adjusting for animals withdrawn from the study for tumor-related reasons, mean group weight loss and survival rates ranging from 87% to 100% were observed, indicating that the investigational drug was well tolerated.

[0355] As shown in Table 8, the efficacy experiment was established with 8 groups of 15 mice each, and each mouse was treated with three doses of Composition 002 alone or in combination with anti-mPD-1. The tumor volume range at the beginning of the experiment was 50 mm 3 Up to 150 mm 3 .

[0356] Table 8: Design of in vivo efficacy experiments

[0357] Composition 002 vehicle: 0.9% NaCl; Anti-mPD-1 vehicle: PBS

[0358] When it reaches more than 1,500 mm in the first animal 3 The experiment was concluded at the end of the experiment when the tumor volume reached the termination standard of . The experiment was divided into two groups, groups 2 to 7 ended on day 8, and groups 1 and 8 ended on day 10.

[0359] Tumor samples collected at the end of the study were used for downstream analysis of tumor infiltrating leukocytes (TILs). Tumors from 10 animals in each group were evaluated by FC analysis using two predefined marker panels. and cells, Tregs, granulocyte-MDSCs, monocyte-MDSCs, NK cells, and M1 / M2 macrophage populations.

[0360] EDTA plasma samples collected 3 days after treatment initiation and at treatment termination, as well as tumor samples, were analyzed using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A to assess changes in various cytokines.

[0361] Table 9 is a summary of the samples collected in this study.

[0362] Table 9: Sample Collection

[0363] Endpoint plasma samples were divided into 70 μl aliquots and the remaining

[0364] The anti-tumor 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 the minimum T / C value expressed as a percentage.

[0365] A summary of vaccination and randomization data is given in Table 10. Individual dosing schedules, efficacy, body weight, and survival data are given in Tables 11 and 12. Tumor growth curves and FC data are shown in Figures 22 to 24.

[0366] Table 10: Experimental Overview

[0367] 1 Random grouping range [mm 3 ]

[0368] Table 11: Anti-tumor efficacy

[0369] n / a = not applicable; nr = not reached (i.e., group median RTV was always < 200% / 400%)

[0370] Efficacy rating: + + + +: T / C < 5%; + + +: 5% ≤ T / C < 10%; + +: 10% ≤ T / C< 25%; +: 25% ≤ T / C < 50%; + / : 50% ≤ T / C ≤ 65%; : T / C > 65%

[0371] ¹ Vehicle for Compound 002: 0.9% NaCl; Vehicle for anti-mPD-1: PBS; ² Minimum T / C value is calculated based on average RTV value.

[0372] Table 12: Weight loss and survival rate

[0373] Vehicle for Composition 002: 0.9% NaCl; Vehicle for anti-mPD-1: PBS.

[0374] 1 The time of minimum mean weight was recorded when at least 50% of the group remained; nr: not relevant, no weight loss was recorded (i.e., group mean RBW was always > 100%).

[0375] 2 The ratio of the number of animals in the group that survived until the last day of the experiment to the total number of animals in the group.

[0376] 3 Adjusted survival rates are for (i.e., include) all animals euthanized and sample collected for tumor-related reasons.

[0377] Materials and methods

[0378] Test agent

[0379] Unless otherwise stated, 1 The amount of active pharmaceutical ingredient delivered

[0380] deal with

[0381] formula

[0382] Anti-mPD-1 vehicle: PBS carrier

[0383] Vehicle for Composition 002: 0.9% NaCl

[0384] Test agents were dissolved in or diluted with appropriate vehicles on dosing days as indicated in the table below.

[0385]

[0386] All dosing solutions were administered in a dose volume of 10 ml / kg.

[0387] animal

[0388] Animals (female C3H / HeNCr1 mice) were shipped from Charles River at a standard age of 4 to 6 weeks and were allowed a minimum of one week of acclimatization upon arrival before use. Animals were arbitrarily numbered using radio frequency identification transceivers (Planet ID) during tumor implantation. Each cage was labeled with a record card indicating all relevant experimental details.

[0389] Animals were housed individually in ventilated cages (TECNIPLAST Sealsafe-IVC-System, TECNIPLAST, Hohenpeissenberg, Germany) in Type III or Type II long cages, depending on group size. They were maintained under a 14L:10D artificial light cycle. Cage temperature was maintained at 22 ± 1°C, relative humidity was 40% to 70%, and air changes were 60 to 65 times / hour. Dust-free bedding consisting of poplar sawdust (ABEDD, LAB & VETService GmbH, Vienna, Austria, product code: LTE E-001) measuring approximately 5 mm × 5 mm × 1 mm and additional nesting material was used. Cages, including bedding and nesting material, were changed weekly. Animals were fed autoclaved, global, 19% protein extruded rodent chow from Envigo RMS SARL and sterile-filtered, acidified (pH 2.5) tap water, with feed and water changed twice weekly. Feed and water were available ad libitum. All materials were autoclaved before use.

[0390] If necessary, add nutrient-enhanced hydrogel (from Maine, USA) DietGelRecovery nutritional recovery gel from the company was provided to the animal cage and replaced every other day.

[0391] Tumor cell culture and implantation

[0392] The bladder tumor xenografts used in this study were derived from the commercially available cell line MBT-2.

[0393] Cells were grown 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) at 37°C in a humidified atmosphere with 5% CO2 and passaged at 40% to 60% density using TrypLE Express (Thermo Fisher, #12605-010). Animals were anesthetized by inhalation of isoflurane and received 1 × 106 Tumor cells (100 μl suspension in PBS) were counted. Cell viability in the cell suspension before and after tumor inoculation was determined using a CASY TT cell counter (OLS OMNI LifeScience GmbH & Co. KG, Bremen, Germany).

[0394] Enrollment / start of experiment

[0395] Animals were monitored until tumor engraftments reached 50 mm in a sufficient number of animals. 3 Up to 150 mm 3 The study volume standard was used. Mice were grouped according to comparable group median and mean tumor volumes. In this report, the grouping process (enrollment, stratified randomization) is referred to as randomization. The date of randomization is designated as day 0 of the experiment.

[0396] The time from implantation to randomization at the desired tumor volume is expressed as "induction time (IT)" days.

[0397] Animal monitoring

[0398] Routine monitoring of animals was performed at least twice daily on weekdays and at least daily on weekends and public holidays. Routine monitoring included inspection of deceased animals, assessment of animal welfare and tumor growth through observation, and control of feed and water supply and technical housing conditions. Any observed or suspected compromise of animal welfare was recorded. Observations and possible outcomes, such as application of euthanasia criteria or veterinary care measures, are reported in Table 12. Autopsies of animals were performed when deemed necessary.

[0399] weight

[0400] The animals were weighed daily for the first week and then three times a week, or daily if weight loss of more than 10% was recorded. The relative weight of individual animals was calculated by dividing the individual weight on day x (BW X ) divided by the individual body weight on the day of randomization (BW0) and multiplied by 100:

[0401] For evaluation purposes, group mean relative body weight (RBW) was calculated. As long as at least 50% of the animals in the group remained alive, the group mean RBW value was used to enter data in the table.

[0402] The individual weight change % was calculated by calculating the weight change (BW x -BW 0) Divide by the body weight on the day of randomization (BW0) and multiply by 100 to calculate:

[0403] Tumor volume

[0404] Absolute tumor volume (ATV) was measured in two dimensions using a digital caliper (S_Cal EVO Bluetooth, Switzerland) on the day of randomization and then three times a week. Tumor volume was calculated according to the following formula: Tumor volume = (1 × W 2 )× 0.5 Where l = maximum diameter, and w = tumor width (perpendicular diameter) (mm).

[0405] The relative volume of the individual tumor on day x (individual RTV) was calculated by dividing the absolute individual tumor volume on day x (T x ) divided by the absolute individual tumor volume of the same tumor on the day of randomization (T0) multiplied by 100:

[0406] As long as at least 50% of the animals in the group remain alive, the group mean RTV value can be used to construct growth curves and for treatment assessments.

[0407] To calculate the mean tumor volume for the group, values from all animals alive on the date in question were included.

[0408] Administration of treatment

[0409] Dosing was performed as described in Table 11. Subcutaneous treatments were applied to the loose skin of the flank rather than the usual loose skin of the neck to facilitate post-treatment blood collection.

[0410] The first day of dosing was the day of randomization (Day 0).

[0411] Dosage adjustments

[0412] When substantial weight loss is documented in efficacy studies, the following measures are taken: Individual animals with > 10% body weight loss were weighed daily. Individual animals that lost >15% of their body weight were not treated.

[0413] Provide access to feed and water for animals that have lost > 10% of their body weight.

[0414] Dosing was resumed when individual animals recovered ≥ 90% of their RBW.

[0415] Note: If any animal in a group / cage requires assistance in obtaining feed and water, DietGel should be provided to all animals in the group / cage. Omission of dosing may also be applied under the guidance of the responsible veterinarian to minimize any other impairment of animal welfare.

[0416] Euthanasia standards

[0417] In accordance with animal welfare regulations and the relevant SOPs of Charles River Discovery Research Services Germany, the following humane endpoints were applied to individual animals, regardless of experimental status: Tumor volume > 1500 mm 3 (1200 mm before the weekend 3 ) Tumors that ulcerate or penetrate the skin Skin necrosis at the tumor site > 5 mm to 8 mm in diameter Weight loss > 30% on any one measurement day Sustained weight loss > 20% for more than two days Rapid weight loss > 20% within two days Severe impairment of overall condition (apathy, pain, markedly decreased food and water intake, difficulty breathing, unusual habits or behaviors) In cases where individual animals met the criteria for euthanasia, sampling was performed before the scheduled time and, if applicable, at the correct time interval after administration of the last administrable dose.

[0418] Tumor samples

[0419] Tumors were collected immediately after euthanasia and divided into three parts when possible. One third was prepared for FC analysis. The second part was quickly frozen in liquid nitrogen for cytokine analysis, and the third part was transferred to a fixative (FFPE samples).

[0420] Fixation was performed in 10% neutral buffered formalin for approximately 24 hours. The fixative was then changed by immersing the specimen in 70% ethanol for up to 7 days. The specimens were then dehydrated by sequential incubation in the following solutions: 70% ethanol (twice for 0.5 hours), 80% ethanol (twice for 1 hour), 100% ethanol (twice for 0.5 hours), 100% isopropanol (1.5 hours), and xylene (twice for 1 hour and 1.5 hours). Finally, the specimens were paraffin-infiltrated and embedded.

[0421] Tumor samples were not collected in cases of complete remission or severe ulceration of the tumor site.

[0422] For FC analysis, tumors were cut into 2 mm to 4 mm slices and processed using the Miltenyi Mouse Tumor Dissociation Kit according to the manufacturer's instructions. Briefly, tumor pieces were incubated with the provided enzyme mix on a gentleMACS dissociator, the resulting cell suspension was filtered through a MACS Smart filter (100 μm; Miltenyi, #130-110-917), the cells were centrifuged at 300 × g for 5 minutes, and the supernatant was discarded.

[0423] Resuspend the cells in 1× ACK lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.2 to 7.4) and incubate at room temperature for 1 to 3 minutes. Pellet the cells by centrifugation at 300 × g for 5 minutes and remove the supernatant. Wash the cells by resuspending them in FC buffer (2% FBS in PBS) and centrifuging them at 300 × g for 5 minutes. Remove the supernatant, resuspend the cells in FC buffer, count the cells, and process them for FC analysis at 5 × 10 cells per well. 6 cells.

[0424] blood sample

[0425] Blood was collected by retrobulbar sinus puncture under isoflurane anesthesia.

[0426] Plasma was prepared by collecting blood directly on ice into standard plasma vials containing EDTA as an anticoagulant, followed by centrifugation at 2000 × g for 5 minutes at 4°C. Plasma was transferred to fresh tubes on ice, and samples were stored at -80°C until analysis or transport.

[0427] Flow cytometry

[0428] Antibody Series A

[0429] 1 Intracellular markers

[0430] Antibody Series B

[0431] 1 Intracellular markers

[0432] The flow cytometry (FC) buffer used was PBS containing 2% FBS. The FC blocking antibody used was purified CD16 / 32 (2.4G2), 0.5 mg / ml (#553142 - BD Biosciences).

[0433] The cells were transferred to 96-well plates (5 × 10 6 cells / well). The cells were pelleted by centrifugation at 400 × g for 5 minutes, and the supernatant was removed. FC blocking antibody (10 μl / 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 against cell surface markers (see Section 5.3.10.1 for antibodies used; staining for antibody series A and B, respectively) were then added to ZombieAqua Fixable Viability Dye (diluted 1:100 in PBS buffer) as recommended by the manufacturer, and the plate was incubated at 4°C in the dark for 30 minutes. The cells were washed by adding 200 μl of FC buffer, followed by centrifugation at 400 × g for 5 minutes, and the supernatant was removed.

[0434] To stain intracellular mouse FoxP3, add 200 μl of fixative solution (BD Pharmingen #519006124) to the relevant wells and incubate the plate at 4°C in the dark for 30 minutes. Pellet the cells by centrifugation at 400 × g for 5 minutes at room temperature and remove the fixative solution. Wash the cells by resuspending them in 200 μl of permeabilization solution (BD Pharmingen #519006125) prewarmed to 37°C and centrifuging the plate at 400 × g for 5 minutes, carefully removing the permeabilization solution. Add fresh permeabilization solution (200 μl) to the relevant wells and incubate the plate at 37°C in the dark for 30 minutes. Pellet the cells by centrifugation at 400 × g for 5 minutes and discard the permeabilization solution. Wash the cells once in 200 μl of FC buffer and incubate with FoxP3 antibody in 40 μl of FC buffer per well. After incubation at room temperature in the dark for 20 minutes, 200 μl of FC buffer was added, the plate was centrifuged at 400 × g for 5 minutes, and the FC buffer was removed.

[0435] Finally, cells were resuspended in 200 μl of FC buffer, transferred to a deep-well plate supplemented with 200 μl of FC buffer, and analyzed using an Attune NXT acoustic focusing flow cytometer (violet (405 nm) / blue (488 nm) / yellow (561 nm) / red (638 nm) laser configuration).

[0436] Data evaluation

[0437] Survival rate

[0438] Survival rate (Table 12) is calculated by counting the number of animals that survived to the last experimental day in each group and dividing them by the total number of animals in the group. Except for the end point of collecting samples and groups, animals that died or euthanized on the last day of the group for any other reason are not considered survivors. The adjusted survival rate in Table 2 is calculated by counting all surviving animals (including animals euthanized due to tumor-related reasons) and dividing them by the total number of animals in the group. Euthanasia for the following reasons is classified as tumor-related: 1) tumors that meet the volume-related euthanasia criteria, including paratumors, and 2) ulcerated tumors. Animals euthanized due to cachexia symptoms induced by tumors are not considered to be tumor-related.

[0439] Tumor volume doubling / quadrupling time

[0440] The doubling and quadruple times (Td, Tq) of the tumor volume in the test and control groups were defined as the time interval (in days) required for the group to reach 200% or 400% of the median RTV. The data are listed in Table 11.

[0441] Inhibition of tumor growth, % of test / control value (minimum T / C value)

[0442] The test value on a specific day is compared with the control value (T / C 平均 %) based on the test group on day x compared to the control

[0443] The ratio of the mean RTV values of the groups was multiplied by 100 for calculation.

[0444]

[0445] The minimum (or best) T / C recorded for the experimental group during the experiment 平均 The value represents the maximum antitumor efficacy of the corresponding treatment. Note that the minimum T / C 平均 values, including any values produced by using the LOCF method.

[0446] Use the group minimum T / C as follows 平均 Values for efficacy rating:

[0447] Flow cytometric analysis

[0448] Flow cytometry data were analyzed using FlowJo data analysis software. The software automatically determined the subpopulation frequency relative to the total population expressed as a percentage. FC results were expressed as the percentage of the corresponding total population and the total count of each population. Doublet exclusion was performed to include only single cells compared to the forward scatter area according to the forward scatter height, followed by forward / side scatter to determine the leukocyte gate and distinguish live cells / dead cells. Fluorescence minus one (FMO) control was used to establish the correct gate. Further gates were set as needed to assess the specified population. The population information analyzed is shown in the table below.

[0449] Series A, T cells, MDSC

[0450] Series B, NK cells, macrophages

[0451] Statistical analysis

[0452] To evaluate the statistical significance of the antitumor efficacy, the nonparametric Kruskal-Wallis test [1] was performed, followed by the Dunn's multiple comparison method [2].

[0453] Individual ATVs were compared between the test and control groups on the final day of the study, when all groups were available. Statistical analysis was performed only when at least 50% of the initially randomized animals remained in the relevant group. No statistically significant differences in tumor volume were observed between the control and test groups.

[0454] For FC data, Kruskal-Wallis test / Dunn post hoc test was performed to compare the percentages of test and control groups for different cell populations. FIG. 23A to FIG. 23B Statistically significant differences between the test and control groups are marked in .

[0455] All p values < 0.05 were considered statistically significant. Statistical calculations were performed using R (version 3.1.0; https: / / www.r-project.org / ), with Kruskal-Wallis and Dunn post hoc tests according to Hollander and Wolfe [3] or GraphPad Prism bioanalysis software (GraphPad software version 9.0 for Microsoft Windows, San Diego, CA, USA, https: / / www.graphpad.com / ).

[0456] Results and Discussion

[0457] Anti-tumor efficacy

[0458] In this study, the anti-tumor efficacy of Composition 002 and anti-mPD-1 was evaluated in a C3H mouse syngeneic MBT-2 tumor model. An experimental overview is given in Table 10. The efficacy results are summarized in Tables 11 and FIG. 22A to FIG. 22B middle.

[0459] Monotherapy with 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of Composition 002 did not show antitumor activity against the MBT-2 tumor model in this study (all minutes, T / C values = 100%).

[0460] Monotherapy with 5 mg / kg anti-mPD-1 did not show antitumor activity against the MBT-2 tumor model in this study (minutes, T / C value 100%).

[0461] In this study, 0.5 mg / kg, 1 mg / kg, and 2 mg / kg of Composition 002 in combination with anti-mPD-1 were also ineffective against the MBT-2 tumor model (minimum T / C value ≥ 71.6%).

[0462] On day 7 of the experiment, the last day for all groups under study, no statistically significant differences in tumor volume were observed between the test groups and the vehicle control group.

[0463] Flow cytometric analysis

[0464] FC analysis was performed on cells isolated from MBT-2 tumors at the end time point of day 10 (Groups 1 and 8) or day 8 (Groups 2 to 7). FC results are shown in Figures 23A to 23F (percentage) and 24A to 24F (Cell counts) were performed. The main observations are listed below.

[0465] In all tested groups of tumors, CD45 + The percentage of cells was lower than that of the control group ( Figure 23A This difference was statistically significant for the 2 mg / kg Composition 002 / anti-mPD-1 and 1 mg / kg Composition 002 / anti-mPD-1 groups when analyzed using Series A antibodies. Similar results were obtained using Series B antibodies, but significant differences were also shown for the 1 mg / kg and 0.5 mg Composition 002 monotherapy groups.

[0466] Compared with the control group, all test groups except the 0.5 mg / kg Composition 002 / anti-mPD-1 group showed The frequency of cell population increased significantly. + and CD8 + The intra-group variability in the percentage of CD4 cells was very high and was only observed in the 2 mg / kg Composition 002 monotherapy group. + There was a significant difference in Treg cells, with frequencies statistically significantly lower compared to controls. Tregs were significantly increased in the three Composition 002 monotherapy groups and the anti-mPD-1 monotherapy group, as well as the 1 mg / kg Composition 002 / anti-mPD-1 group ( Figure 23B ).

[0467] The percentage of granulocyte MDSCs was significantly lower in all tested groups except the 2 mg / kg Composition 002 monotherapy and 0.5 mg / kg Composition 002 / anti-mPD-1 groups, whereas the percentage of monocyte MDSCs was significantly higher in all tested groups except the 2 mg / kg Composition 002 monotherapy and 0.5 mg / kg Composition 002 / anti-mPD-1 groups ( Figure 23C ).

[0468] For NK cells, no significant difference was observed between the test and control groups ( Figure 23E ). The intra-group variability of the experimental group was higher than that of the control group.

[0469] F4 / 80 in all test groups except the 0.5 mg / kg Composition 002 / anti-mPD-1 group + The frequency of M1 macrophages in the cells was significantly higher, with F4 / 80 in all tested groups except the 0.5 mg / kg Composition 002 / anti-mPD-1 group. + The frequency of M2 macrophages in the cells was significantly lower ( Figure 23F ).

[0470] Body weight change, survival, and observational outcomes

[0471] The results are summarized in Tables 12 and Figure 25 middle.

[0472] Minimal body weight loss (BWL) was observed in this study, indicating that the test drug was well tolerated. A maximum 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 died on day 8 in the 0.5 mg / kg Composition 002 monotherapy group, and two animals died on days 7 and 10 in the 0.5 mg / kg Composition 002 / anti-mPD-1 group, resulting in an adjusted survival rate of 87% for animals euthanized for tumor-related reasons. One animal each died on days 6 and 5 in the other two combination groups (2 mg / kg Composition 002 / anti-mPD-1 and 1 mg / kg Composition 002 / anti-mPD-1), resulting in an adjusted survival rate of 93%. Survival in the remaining groups was 100%.

[0473] References

[0474] [1] Kruskal WH, Wallis WA: Use of Ranks in One-criterion VarianceAnalysis. J. Am. Stat. Assoc. 1952, 47: 583-621.

[0475] [2] Dunn OJ: Multiple Comparisons Using Rank Sums. Technometrics,1964, 6(3), pp. 241-252.

[0476] [3] Hollander M, Wolfe DA: Nonparametric Statistical Methods. NewYork: John Wiley & Sons, 1973, Pages 115–120.

[0477] Acronyms and abbreviations:

[0478] Example 3: Preparation of a lyophilized composition for injection containing non-activated Streptococcus pyogenes

[0479] Streptococcus pyogenes (A group, 3 types, Su bacterial strain) is cultivated in appropriate culture medium. After appropriate incubation period, by centrifugal collection bacterium, it is washed, resuspended, and with hydrogen peroxide treatment to kill bacteria. Killed bacteria are centrifuged, washed and resuspended in suspension medium (such as Berheimers basal medium, BBM) and filtered. With benzylpenicillin-treated bacterial suspension, and heated at 37 ℃ for about 10 to 45 minutes, at 45 ℃ for about 20 to 60 minutes. Prepare final large volume suspension. Fill bottle and lyophilize with final large volume suspension. The quantitative formula of the different suggested dosage strengths of the exemplary composition comprising non-activated Streptococcus pyogenes is provided in Table 13A. These compositions are based on lyophilized product. Before lyophilization, all bottle strengths were filled with the suspension of same volume (0.41 mL).

[0480] Table 13A: Quantitative formulations of various dosage strengths of exemplary composition 002

[0481] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005 mg / mL to 0.01 mg / mL. The volume of product delivered to a subject at this concentration can vary.

[0482] Table 13B: Examples of Excipients for Exemplary Compositions After Suspension in 0.9% Saline

[0483] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005 mg / mL to 0.01 mg / mL. The volume of product delivered to a subject at this concentration can vary.

[0484] Example 4: Compositions containing non-activated Streptococcus pyogenes activate T cells

[0485] T cells were isolated from peripheral blood mononuclear cells (PBMCs - two healthy donors) using RapidSheres magnetic beads. T cells (500,000) were seeded in 96-well plates. T cells were treated with 0.2 KE / mL and 0.8 KE / mL of composition 002 for 72 hours. T cells and supernatants were collected and analyzed for immune checkpoint biomarkers and cytokines by FACS and ELISA, respectively (see table below).

[0486]

[0487] like Figure 26 As shown, treatment with Composition 002 did not alter CD4 + and CD8 +The number of T cells. Composition 002 induces CD4 + T cells ( Figure 27A ) and CD8 + T cells ( Figure 27B ) and the expression of immune checkpoint molecules CTLA4, PD-1, LAG3, TIM3 and TIGIT in the human breast cancer cell line.

[0488] Example 5: In vivo efficacy of non-viable Streptococcus pyogenes cells as monotherapy and in combination with anti-mPD-1 in an orthotopic bladder cancer model (MB49 bladder cancer cells)

[0489] The anti-tumor efficacy of a lyophilized formulation of Streptococcus pyogenes (Group A, Type 3, substrain) treated with penicillin alone and in combination with an antibody targeting programmed cell death protein 1 (PD-1) was evaluated in a mouse orthotopic bladder tumor model (MB49 bladder cancer cells). The study design for monotherapy is shown in Table 6 below. Animals were randomly assigned to study groups by tumor-associated bioluminescence. A lyophilized formulation of a penicillin-treated Streptococcus pyogenes composition was administered once a week for 4 weeks. Mice were observed for 1 week after treatment. Mice were examined daily for 5 days a week. Body weight was measured twice a week. Bioluminescence imaging (BLI) was obtained 1 to 2 times a week during the in vivo phase for 4 weeks. The optimal dose of a lyophilized formulation of Streptococcus pyogenes treated with penicillin for combined study with an anti-PD-1 antibody was selected.

[0490] Table 14: Study design for the treatment of an orthotopic bladder tumor model (MB49) in mice using a lyophilized formulation of penicillin-treated Streptococcus pyogenes

[0491] The study design for the combination therapy is shown in Table 15 below. Animals were randomized to study groups based on tumor-associated bioluminescence. A lyophilized formulation of the penicillin-treated Streptococcus pyogenes composition was administered once weekly for 4 weeks. Mice received anti-PD-1 antibody twice weekly. Mice were observed for 1 week after treatment. Mice were examined daily for 5 days per week. Body weight was measured twice weekly. Bioluminescence imaging (BLI) was obtained 1 to 2 times per week during the in vivo phase for 4 weeks.

[0492] Table 15: Study design for the treatment of an orthotopic mouse bladder tumor model (MB49) with a lyophilized formulation of penicillin-treated Streptococcus pyogenes and anti-PD-1 antibodies

[0493] Example 6: Preparation of a lyophilized composition for injection containing non-activated Streptococcus pyogenes

[0494] Streptococcus pyogenes (A group, 3 types, Su bacterial strains) is cultivated in appropriate culture medium. After appropriate incubation period, by centrifugal collection bacterium, it is washed, resuspended, and killed bacterium with hydrogen peroxide treatment. Killed bacterium is centrifuged, washed and resuspended in suspension medium (such as Berheimers basal medium, BBM) and filtered. Treat bacterial suspension with benzylpenicillin, and heat about 10 to 45 minutes at 37 ℃, and about 20 to 60 minutes at 45 ℃. Prepare final large volume suspension. Fill bottle and lyophilize with final large volume suspension. The quantitative formula of different suggested dosage strengths of the exemplary composition comprising non-activated Streptococcus pyogenes is provided in table 16. These compositions are based on lyophilized product. Before lyophilization, all bottle strengths were filled with suspension of same volume (0.41mL).

[0495] Table 16: Quantitative formulations of various dosage strengths of exemplary compositions

[0496] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005 mg / mL to 0.01 mg / mL, at which concentration the volume of product delivered to the subject can be varied. Table 17 provides a quantitative formulation of an exemplary composition suspended in 0.9% saline at a final cell concentration of 0.01 mg / mL.

[0497] Table 17: Examples of excipients for exemplary compositions after suspension in 0.9% saline

[0498] For administration, the lyophilized powder is suspended in isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005 mg / mL to 0.01 mg / mL. The volume of product delivered to a subject at this concentration can vary.

[0499] Example 7: In vitro and in vivo efficacy of non-viable Streptococcus pyogenes cells as monotherapy and in combination with immune checkpoint inhibitors

[0500] Materials and methods: Immunogenic cell death To evaluate the release of damage-associated molecular pattern molecules (DAMPs), bladder tumor MB49 cells were seeded in 96-well plates (2 × 10 4Three replicates were set up for each experimental point and incubated in high-glucose, phenol red-free DMEM medium containing HEPES (Thermostat, catalog number: 21063029), 10% heat-inactivated fetal bovine serum (HI FBS) (Seradigm Avantor, catalog number: 1500-500H), and 1% penicillin / streptomycin (P / S) (Gibco-ThermoFishe, catalog number: 15140-122) at 37°C and 5% CO2 for 24 hours.

[0501] Cells were treated with 0 KE / mL, 0.2 KE / mL, 0.8 KE / mL, 3.2 KE / mL, and 12.8 KE / mL (1 KE = 0.1 mg) of composition 002 for 24 hours or 1 μM mitoxantrone (Sigma, catalog number: M6545) as a positive control. After treatment, the plates were centrifuged at 400 g for 5 minutes, and the supernatant was collected for HMGB1 quantification, while the cells were collected for flow cytometry analysis.

[0502] HMGB1 was quantified using the Promega Lumit Immunoassay (Promega, catalog number: W6110) kit according to the manufacturer's instructions. The mean RLU of the appropriate background control (medium alone, or medium alone treated with the corresponding concentration of drug) was subtracted from the data for each triplicate treated cell sample. Fold induction of the treated sample was calculated according to the following equation: (RLU of treated cells – RLU of cell-free medium + drug) / (mean RLU of untreated cells – RLU of cell-free medium). Annexin V-FITC (Abcam, catalog number: Ab14085) and Calreticulin-AF700 (R&D Systems, catalog number: IC38981N) markers were quantified by flow cytometry. Briefly, cells were stained with Calreticulin-AF700 for 30 minutes at 4°C, washed, and resuspended in binding buffer from the Annexin V kit (Abcam, catalog number: Ab14085). Annexin V and 50 μg / mL propidium iodide (Abcam catalog number: Ab14085) were added to the solution and incubated at 25°C in the dark for 5 minutes.

[0503] For exogenous ATP analysis (eATP), RealTimeGlo eATP detection reagent (Promega, catalog number: GA5010) was added to the culture medium before composition 002 treatment, and eATP luminescence measurements were performed 24 hours after treatment. After the final measurement, 400 μg / mL Digitonin (Promega, catalog number: G9441) was added to the culture medium to assess total ATP and overall cell health as an assay control. eATP was calculated using the following formula: [(average of triplicate treated cell RLUs – average of cell-free medium + drug RLUs) / (average of untreated cell RLUs – cell-free medium RLUs) x 100 – 100], where RLU represents luminescence after background removal.

[0504] Dendritic cell activation and phagocytosis assay

[0505] Bone marrow cells were collected by flushing the femur with RPMI 1640 culture medium (ATCC, catalog number: 30-2001). Disaggregated cells were filtered twice through a 70 μM pre-wetted filter to remove cell clumps and counted with 3% acetic acid and methyl blue (StemCell Technologies, catalog number: 07060). Bone marrow cells were then resuspended in RPMI 1640 culture medium supplemented with 2 mM GlutaMAX (ThermoFisher, catalog number: 30-2001), 10% HI FBS (Avantor, catalog number: 1500-500H), 1% P / S (Gibco, catalog number: 15140-122), 50 ng / ml GM-CSF (PeproTech, catalog number: 300-03), 25 ng / mL of IL-4 (PrepoTech catalog number: 200-04), and cultured at 37°C and 5% CO in 96-well plates (3 × 10 4 Cells were cultured for 48 h in a 400 μg / well culture medium. After 24 h of culture, half of the culture medium was replaced, and dendritic cell (DC) differentiation was allowed to continue. Subsequently, the culture medium was completely replaced, and the cells were cultured for an additional 72 h to complete DC differentiation.

[0506] MB49 cells (2.5×10 6Cells (cells) were cultured in T-25 culture flasks in DMEM high glucose medium containing HEPES (ThermoFisher, catalog number: 12430054), 10% HI FBS, and 1% P / S, and treated with composition 002 (0 KE / mL, 0.05 KE / mL, 0.2 KE / mL, 0.8 KE / mL, 1.6 KE / mL, and 3.2 KE / mL) for 24 hours. Denazolidinone (1 μM) (Tocris R & D, catalog number: 7336) was used as a positive control. After treatment, the cells were resuspended and washed to remove composition 002 and treated with Vybrant TM The cells were stained with DiO cell labeling solution (ThermoFisher, catalog number: V22886) at 37°C for 20 minutes.

[0507] Pre-labeled MB49 cells and DCs were cultured at a 2:1 ratio (3 × 10 4 DC: 1.5×10 4 MB49 cells) were co-cultured for 24 hours. DCs were identified using CD80-PE (BioLegend, catalog number: 305207), CD86-BV421 (BioLegend, catalog number: 305425), CD11c-AP (BioLegend, catalog number: 337207), and HLA-DR-BUV395 (BD Bioscience, catalog number: 565972). Pre-labeled MB49 cells were identified using DiO-FITC, and overall cell viability was analyzed by flow cytometry using APC-eFluor780 (eBioscience catalog number: 65-0865-14).

[0508] Cytotoxicity and cytokine release of composition 002

[0509] Bladder cancer RT112 cells were cultured in EMEM culture medium (ATCC, catalog number: 30-2003) supplemented with 2 mM GlutaMax (ThermoFisher, catalog number: 35050061), 1% NEAA (ThermoFisher, catalog number: 11140050), 10% HI FBS and 1% P / S. 5637 cells were cultured in RPMI 1640 culture medium (ATCC, catalog number: 30-2001) supplemented with 2 mM GlutaMax, 10% HIFBS and 1% P / S. Both cell lines were pre-labeled at 37°C using CellTracker Red solution (1 μM) (Invitrogen, catalog number: C34552) for 30 minutes. Then, the cells were washed and plated in 96-well plates (2.5 × 10 4 The cells were cultured at 37°C and 5% CO2 for 24 hours.

[0510] Fresh peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using the EasySep Human PBMC Direct Isolation Kit (Stem Cell Technologies catalog number: 19654) supplemented with 6 mM EDTA (ThermoFisher, catalog number: 15575-038) according to the manufacturer's instructions. PBMCs were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P / S.

[0511] The cells were cultured alone or in combination with PBMCs (1.65 × 10 5 cells / well) co-cultured tumor cells (2.5×10 4Cells / well) were treated with composition 002 (0.2 KE / mL) for 75 h. After treatment, the plate was centrifuged at 400g for 5 minutes and the supernatant was collected. Inflammatory cytokines were stained using V-Plex Human Proinflammatory Factor Panel 1 (Meso Scale, catalog number: K15049D-1) and detected using a plate reader. Then, the cells were washed and trypsinized for flow cytometry analysis. Tumor cell viability was quantified using CellTracker Red-PE (Invitrogen, catalog number: C34552), Live / DeadDye-efluor780 (eBioscience, catalog number: 65-0865-14) and CD45-BUV395 (BD, catalog number: 563792) and PBMC were excluded from the analysis.

[0512] T cell activation

[0513] Frozen PMBCs were thawed and slowly dissociated by resuspending the cells in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S with 100 μg / mL DNase I (StemCell Technologies, catalog number: 17951) and incubated at 25 o Incubate at 4°C for 15 minutes. EasySep buffer (StemCell Technologies, catalog number: 20144) was added to the mixture, and the cells were filtered through a 37 µM cell strainer (StemCell Technologies, catalog number: 07900). T cells were isolated using the EasySep Human T Cell Isolation Kit (StemCell, catalog number: 17951) according to the manufacturer's instructions.

[0514] Cells were plated in 96-well plates (5 × 10 5 / well) and treated with composition 002 (0.2 KE / mL) for 72 hours at 37°C, 5% CO2. The plate was centrifuged and the supernatant was collected and IFN-g (Invitrogen, catalog number: BMS228) and granzyme B (Invitrogen, catalog number: BMS2027-2) were quantified by ELISA. T cells were collected and stained for the following markers: CD4-BUV395 (BD, catalog number: 564724), FoxP3-PE (BD, catalog number: 560852); CD8-FITC (BioLegend catalog number: 344704), PD-1-PE-Cy7 (BioLegend, catalog number: 329918), CTLA-4-BV421 (BioLegend, catalog number: 369606), TIGIT-BV711 (BioLegend Cat. No. 372742), TIM3-BV605 (BioLegend, catalog number: 345018), LAG3-APC (BioLegend, catalog number: 369212), Ki67-efluor506 (eBioscience, catalog number: 69-5698-82), Live / Dead dye-efluor708 (eBioscience, catalog number: 65-0865-14). Data were acquired by flow cytometry.

[0515] PD-L1 analysis in tumor cells

[0516] 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 in 96-well plates (2.5 × 10 4 cells / well) and grown overnight.

[0517] Frozen PMBCs were thawed and slowly dissociated by resuspending the cells in RPMI-1640 medium supplemented with 10% HI FBS, 1% P / S and 100 μg / mL DNase I, and incubated at 25 o Incubate at 4°C for 15 min. 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, catalog number: 17951) according to the manufacturer's instructions and maintained in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S.

[0518] According to the ratio of 6.6:1 (1.65×10 5 T cells were added to appropriate co-culture wells (100 cells / well) with a 1:1 ratio of 5637 cells to T cells. 5637 cells alone and co-cultured with T cells were treated with Composition 002 for 72 hours at 37°C, 5% CO2. Following treatment, the supernatant was washed and analyzed by flow cytometry for the PD-L1 (BioLegend, Catalog No. 374510) marker in pre-labeled tumor cells.

[0519] In vitro cytotoxicity analysis using the xCELLigence Real-Time Cell Analyzer (RTCA)

[0520] Fresh peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using 2x EasySep human PBMC direct isolation kit (Stem Cell Technologies, catalog number: 19654) and supplemented with 6 mM EDTA (ThermoFisher, catalog number: 15575-038) according to the manufacturer's instructions. PBMCs were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P / S. 5637 cells were cultured in RPMI-1640 medium supplemented with 10% HI FBS and 1% P / S and seeded in 96-well E-plates (Agilent, catalog number: 300600910) (5 × 10 5 Cells were plated on a plate for 30 min (10 cells / well) to allow the cells to adhere. After approximately 78 hours, effector cells (human PBMC; effector / target ratio of 6.6:1) were added and treated. Composition 002 (0.8 KE / mL) was treated alone or in combination with the following antibodies for approximately 65 hours in a co-culture environment (tumor cells + PBMC): anti-PD-1 (10 μg / mL-Bioxcell, catalog number: SIM0010), anti-PD-L1 (10 μg / mL-Bioxcell, catalog number: SIM0009), anti-CTLA-4 (10 μg / mL-Selleckchem, catalog number A20001), RecombiMAb IgG4 (Bioxcell, catalog number: CP147) (10 μg / mL), and RecombiMAb IgG4 (Bioxcell, catalog number: CP147) (10 μg / mL) was used as an isotype control. Cell index measurements were collected every 15 minutes by xCelligence RTCA eSight (Agilent) for a total of 143.5 hours (approximately 78 hours of cancer cell culture alone + approximately 65 hours of co-culture). For each well, % cytolysis was calculated using the normalized sample cell index and the normalized average target alone control according to the following formula: % cytolysis = (Cell Index无效应细胞 – Cell Index 效应细胞 ) / Cell Index 无效应细胞 ×100

[0521] MB49 subcutaneous mouse model

[0522] For the dose-finding study, 14-week-old female C57BL / 6 mice were subcutaneously implanted with MB49 bladder cancer cells (2.0 × 10 5 cells / mouse), and 10 mice in each group were randomly divided into groups (80 mm 3 Up to 130 mm 3 Mice were enrolled in groups based on tumor size. Mice were dosed intravenously with Composition 002 (0.08 KE / mouse, 0.4 KE / mouse, and 2 KE / mouse) once weekly for 4 weeks. Tumors were measured twice weekly using a caliper. No humane endpoints (weight loss > 20%, tumor burden > 2000 mm) were achieved during monitoring. 3 Animals with severe edema, open oozing tumor ulcers, severe respiratory distress, severe motor impairment, or loss of righting reflex) were screened for up to 35 days.

[0523] For the combination study, 14-week-old female mice were subcutaneously implanted with MB49 cells (2.0 × 10 5 cells / mouse), and 10 mice in each group were randomly divided into groups (75 mm 3 Up to 130 mm 3 Mice were enrolled at 4 tumor sizes. Composition 002 (0.4 KE / mouse) was administered intravenously once weekly for 4 weeks and / or anti-PD-1 (10 mg / kg) (BioXcell, catalog number: BP0146) was administered twice weekly for 2 weeks. An isotype control (2A3) (BioXcell, catalog number: BP0089) was used as a negative control. Animals that did not reach the humane endpoint were maintained for 5 days after the last dose of Composition 002, then euthanized and their tumors harvested for immunohistochemical analysis.

[0524] EMT6 orthotopic mouse model

[0525] For the dose-finding study, 14-week-old female Balb / c mice were implanted with 5 × 10 5 After 7 days, 6 mice in each group were randomly divided into 50 mm 3 Up to 150 mm 3Tumor size was quantified and each mouse was intravenously administered composition 002 (0.4 KE / mouse, 1 KE / mouse, and 2 KE / mouse) twice a week. Tumor growth and mouse body weight were measured twice a week for 3 weeks. Mice that did not reach the humane endpoint were euthanized on day 35 of the study. Tumors from 3 mice in each group were collected and, after mechanical dissociation, chemically separated by treatment with collagenase D (2.5 mg / mL). Cells were filtered through a 70 μM cell filter and stained for FACS analysis of tumor-infiltrating immune cell populations (Table 18).

[0526] Table 18: FACS analysis of tumor-infiltrating immune cell populations

[0527] result: MB49 bladder cancer cells exposed to Composition 002 underwent apoptosis ( Figure 28A ), accompanied by the production of markers of immunogenic cell death, such as HMGB1, extracellular ATP (eATP), and cell surface expression of calreticulin ( Figures 28B to 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 in turn can effectively activate naive T cells. Since DCs play a key role in recognizing DAMPs associated with immunogenic cell death and subsequent tumor antigen uptake and presentation, the phagocytosis of tumor cells treated with composition 002 by DCs was examined. MB49 cells were treated with composition 002 and then cultured with mouse bone marrow-derived DCs. MB49 bladder cancer cells treated with composition 002 increased phagocytosis of dendritic cells ( Figure 29A ) and phenotypic maturation, as indicated by increased surface expression of CD80, CD86, and HLA-DR ( Figures 29B to 29D Using an in vitro co-culture method of bladder cancer cells (5637 and RT112) and PBMCs, it was found that composition 002 enhanced immune-mediated killing of bladder cancer cells ( FIG. 30A to FIG. 30B Notably, cytokine analysis showed that Composition 002 increased the release of pro-inflammatory Th1 cytokines ( Figure 31A ), creating a favorable environment for inducing cellular and humoral anti-tumor immunity. In addition, Composition 002 also promotes the reduction of Th2 cytokines ( Figure 31B For example, composition 002 reduced the release of IL-10, which is known to inhibit the secretion of various Th1 cytokines by macrophages and dendritic cells.

[0528] To investigate whether Composition 002 induces PD-L1 expression in cancer cells, thereby inhibiting anti-tumor immune responses, bladder cancer cells (5637) in co-culture with PBMCs were treated with increasing concentrations of Composition 002. Composition 002 treatment resulted in increased PD-L1 expression in 5637 cells compared to the control ( Figure 34 Furthermore, stimulation of T cells with Composition 002 alone increased T cell proliferation, as observed by a high percentage of CD4 and CD8 cells showing the KI67 marker ( Figure 32A and Figure 32H In addition, composition 002 promoted the release of IFN-γ and granzyme B ( Figures 33A to 33B ), indicating enhanced cytotoxic activity. However, high levels of LAG3, CTLA4, PD-1, TIGIT, and TIM3 in CD4 and CD8 T cells (respectively) Figure 32B 、 Figure 32C 、 Figure 32D 、 Figure 32E 、 Figure 32F 、 Figure 32J 、 Figure 32I 、 Figure 32K 、 Figure 32L and Figure 32M ) and FOXP3 CD4 + Regulatory T cells ( Figure 32G ) to identify markers of exhausted T cell phenotypes. In summary, the immunostimulatory effects of Composition 002 can be enhanced with additional agents that target these exhausted T cell phenotypes.

[0529] To study the potential synergistic anti-tumor effects of composition 002 with immune checkpoint inhibitors (ICI) anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and anti-PD-1 antibodies in vitro, the xCELLigence platform was selected, which can be used to monitor cell health, proliferation, and cell lysis over time. Target 5637 bladder cancer cells were seeded in a biosensor plate (E-plate) and allowed to attach and proliferate. After 78 hours, fresh PBMCs were added to the top of the 5637 cells and cultured for approximately 65 hours (a total of 143.5 hours) in the presence or absence of composition 002, ICI, or an irrelevant IgG isotype (IgG4 for anti-PD-1, IgG1 for anti-PD-L1 and anti-CTLA4) as a control. When compared to individual treatments, the combination of composition 002 with anti-PD-L1 or anti-CTLA4 in the presence of human PBMCs showed enhanced 5637 cell lysis ( Figures 35A to 35D). It is worth mentioning that the combination of Composition 002 and ICI had a delayed anti-tumor effect compared with Composition 002 alone, indicating that the combined activity was dependent on the expression of immune checkpoints (e.g., PD-L1, CTLA-4) mediated by Composition 002. However, only a slight increase in cell lysis was observed when using anti-PD-1 antibodies compared with Composition 002 alone ( Figures 35E to 35F ).

[0530] Since the response of tumors to immunotherapy (such as anti-PD-1) may depend on the interaction of several cell types in the tumor microenvironment in vivo, the study of the combination of Composition 002 and anti-PD-1 was repeated using a bladder cancer mouse model. Instead of the previously used MBT2 model (Example 2), the MB49 murine bladder cancer model was selected, which has the potential to respond to immune stimulants and is widely used in bladder cancer immunotherapy research. In addition, based on the delayed anti-tumor effects of Composition 002 observed with other ICIs in vitro, mice were treated for three weeks (instead of one week as in the previous in vivo MBT2 bladder cancer study in Example 2). As a monotherapy, Composition 002 effectively reduced tumor growth and increased survival in the MB49 subcutaneous model ( Figures 36A to 36B Further analysis in this same model using Composition 002 in combination with anti-PD-1 showed a reduction in tumor volume compared to Composition 002 or anti-PD-1 as monotherapy ( Figures 37A to 37E For example, on day 20, the mean tumor volumes of the anti-PD-1 alone and combination groups were 264 mm 3 and 159 mm 3 , indicating additional efficacy. Furthermore, 30% complete regressions and 10% tumor-free survivors were observed in the combination group. No complete regressions or tumor-free survivors were observed in the monotherapy group.

[0531] The anti-tumor effect of Composition 002 was also tested in vivo using a murine orthotopic triple-negative breast (TNBC) cancer model (EMT6 cells) characterized by an immunosuppressive tumor microenvironment and resistance to anti-PD-1 therapy. The results confirmed a significant but modest anti-tumor effect of Composition 002 as a single-agent treatment ( Figure 38A ). On immune cells analyzed by flow cytometry, tumor-associated macrophages (TAMs) were identified, with an increase in the anti-tumor macrophage 1 (M1) cell type and a decrease in the pro-tumor macrophage 2 (M2) cell type (i.e., M1-type polarization), which was associated with increased PD-1 expression on T cells ( Figure 38B In the EMT6 TNBC mouse model, systemic delivery of Compound 002 in combination with anti-PD-1 demonstrated significantly superior anti-tumor activity compared to each agent as monotherapy, suggesting a synergistic effect between Compound 002 and anti-PD-1 ( Figures 39A to 39B In addition, by restoring PD-1 levels in T cells, increasing the level of intratumoral CD4 T cells, promoting macrophage polarization toward the M1 phenotype (anti-tumor), and downregulating tumor-promoting Treg cells, Composition 002 combined with anti-PD-1 remodeled the local immune cell population ( Figures 40A to 40C Of particular importance is that Combination 002 combined with anti-PD-1 significantly increased the levels of tumor-infiltrating natural killer (NK) cells compared to the monotherapy groups ( Figure 40D In fact, researchers have already highlighted the advantages of anti-PD-1 / PD-L1 therapy in improving NK cell function and found that blocking the PD-1 / PD-L1 interaction can enhance NK cell killing of tumor cells. In addition, PD-1 / PD-L1 antibodies are completely ineffective in some NK-deficient mouse models.

[0532] 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 mentioned in this specification and / or listed in the application data sheet, including but not limited to U.S. Provisional Patent Application No. 63 / 479,170 filed on January 9, 2023, U.S. Provisional Patent Application No. 63 / 487,224 filed on February 27, 2023, and U.S. Provisional Patent Application No. 63 / 487,232 filed on February 27, 2023, are incorporated herein by reference in their entirety. If necessary, various aspects of the embodiments can be modified to provide additional embodiments using the concepts of various patents, applications, and publications.

[0533] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

Claims

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

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

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

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

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

6. The method of any one of claims 1 to 5, wherein the non-viable S. pyogenes cells 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, 0.5 KE, 1 KE, 2.5 KE, 5 KE, 10 KE, 15 KE, 20 KE, 30 KE, 40 KE, 50 KE, 60 KE, 70 KE, 80 KE, 90 KE, or 100 KE.

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

8. The method according to 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. 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. 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. The method of claim 10, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrilimab, cemiplimab, saxanalizumab, nofalimab, genolizumab, sepalizumab, serotonin, putalizumab, palolizumab, camrelizumab, candurizumab, dotalizumab, penampalizumab, toripalizumab, tislelizumab, sintilimab, or dotalizumab.

12. 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. The method according to claim 12, wherein the PD-L1 antibody comprises atezolizumab, durvalumab, envolimab, sugemalimab, coxilimab, socazolimab, taragolimab, betifisolimab, lesabelimab, pamilizumab or avelumab.

14. 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. 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 PD-L1 inhibitor is an antibody.

16. 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. The method according to claim 16, wherein the CTLA-4 antibody comprises ipilimumab, tesimumab, or tovorelizumab.

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

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

20. The method of any one of claims 1 to 19, wherein the composition comprising non-viable Streptococcus pyogenes cells is administered before, simultaneously with, or after the immune checkpoint inhibitor.

21. The method according to any one of claims 1 to 20, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [Group A, Type 3] Su strain.

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

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

24. The method according to any one of claims 1 to 23, wherein the composition comprising non-viable cells of Streptococcus pyogenes is a lyophilized composition, optionally wherein the lyophilized composition is reconstituted prior to administration.

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

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

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 comprising non-viable cells of Streptococcus pyogenes for use in combination with an immune checkpoint inhibitor to treat triple-negative breast cancer.

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

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

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

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

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

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

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

36. according to the method described in any one in claim 30 to 35, wherein said Streptococcus pyogenes non-viable cells comprise Streptococcus pyogenes Su strain cells.

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

38. The method of any one of claims 30 to 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. 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. The method of claim 39, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrilimab, cemiplizumab, sazanalizumab, tislelizumab, or dotarizumab.

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

43. 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. 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 PD-L1 inhibitor is an antibody.

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

46. 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. The method of claim 46, wherein the CTLA-4 antibody comprises ipilimumab, tesimumab, or tovorizumab.

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

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

50. The method of any one of claims 30 to 49, wherein the composition comprising non-viable Streptococcus pyogenes cells is administered before, simultaneously with, or after the immune checkpoint inhibitor.

51. The method of any one of claims 30 to 50, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [Group A, Type 3] Su strain.

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

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

54. The method of any one of claims 30 to 53, wherein the composition comprising non-viable cells of Streptococcus pyogenes is a lyophilized composition, optionally wherein the lyophilized composition is reconstituted prior to administration.

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

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

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

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

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

60. The method of any one of claims 30 to 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 cancer in situ (CIS) with or without Ta and / or T1.

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

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

64. The method of any one of claims 30 to 62, wherein the subject has received adequate BCG therapy.

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

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

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

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

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