Combination of non-viable cells of Streptococcus pyogenes and immune checkpoint inhibitor for the treatment of triple negative breast cancer and non-muscle invasive bladder cancer

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

1. 11. A method of treating non-muscle invasive bladder cancer in a subject, comprising: (i) administering intravesically to the subject a composition comprising non-viable cells of Streptococcus pyogenes; and (ii) administering to the subject an anti-PD-1 antibody.
2. 22. The method of claim 1, wherein the anti-PD-1 antibody comprises pembrolizumab.
3. 33. The method of claim 1, wherein the subject is a human adult subject.
4. 44. The method of claim 1, wherein the non-muscle invasive bladder cancer is Bacillus Calmette-Guerin (BCG) failed, BCG-unresponsive, BCG-refractory, BCG-relapsing, BCG inadequately treated, the subject has received adequate BCG treatment, or any combination thereof.
5. 55. The method of claim 1, wherein the subject was inadequately treated with BCG.
6. 66. The method of claim 5, wherein the subject did not receive a full BCG dose due to intolerance to or unavailability of BCG drug.
7. 77. The method of claim 1, wherein the anti-PD-1 antibody is administered intravenously.
8. 88. The method of claim 1, wherein the subject has high-grade non-muscle invasive bladder cancer.
9. 99. The method of claim 1, wherein the non-muscle invasive bladder cancer has been identified as high-grade Ta or T1.
10. 1010. The method of claim 9, wherein the subject was diagnosed with recurrent high-grade Ta or T1 disease within 6 months of completion of adequate BCG therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. patent application Ser. No. 19/146,376, filed Jul. 8, 2025, which is a 371 national stage application of International Patent Application No. PCT/US2024/010754, filed Jan. 8, 2024, which claims the benefit of U.S. Provisional Application No. 63/479,170, filed Jan. 9, 2023, U.S. Provisional Patent Application No. 63/487,224, filed on Feb. 27, 2023, and U.S. Provisional Patent Application No. 63/487,232, filed on Feb. 27, 2023, which applications are incorporated herein by reference in their entirety.
BACKGROUND
Triple negative breast cancer refers to breast tumors characterized by the absence of estrogen receptor, progesterone receptor, and HER2. Patients with triple negative breast cancer do not respond to hormonal or trastuzumab-based therapies. Triple negative breast cancer tends to be more aggressive, harder to treat, and more likely to recur than other forms of the disease, such as hormone receptor-positive or HER2-positive breast cancers. Conventional chemotherapy has not been very effective against triple-negative breast cancer, and new treatment options are needed.
Bladder cancer is the tenth most common cancer worldwide, with an incidence rate of almost half a million per year. Non-muscle invasive bladder cancer (NMIBC), which is defined as cancer confined to the bladder mucosa and submucosa, constitutes 75% of bladder cancer cases. The most common histological subtype is urothelial carcinoma. Non-muscle invasive bladder cancer includes papillary tumors within the mucosa (stage Ta), tumors invading the lamina propria (stage T1), and flat high-grade lesions referred to as carcinoma in situ (CIS). NMIBC is primarily managed with local endoscopic/intravesical therapy and surveillance.
Non-muscle invasive bladder cancer's progression risk to muscle invasion or recurrence risk varies according to tumor grade and depth. For instance, at one end of the spectrum, low-grade Ta bladder cancer recurs in almost two-thirds of cases, but seldom (only approx. 6%) progresses into a more invasive disease, whereas NMIBC with high-risk features, including high-grade T1, is reported to have a recurrence rate of almost 50%. Moreover, such cancers progress to invade the muscle in one of five patients, typically within 2 years of diagnosis.
BCG is the standard immunotherapy for NMIBC. Current treatment options following unsuccessful BCG therapy are limited to radical cystectomy the standard of care. For patients who are unfit 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 in BCG-unresponsive disease, particularly considering the recent global BCG shortage, and high morbidity associated with radical cystectomy, there is an unmet need for treatments of high-risk NMIBC.
BRIEF SUMMARY
In one aspect, 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.
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 for treating triple negative breast cancer.
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 for treating triple negative breast cancer.
In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD1/PD-L1/PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.
In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1/PD-L1/PD-L2 axis.
In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [A Group, Type 3] Su strain.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes. 
In another aspect, 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.
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 for treating non-muscle invasive bladder cancer.
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 for treating non-muscle invasive bladder cancer.
In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD1/PD-L1/PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA 4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.
In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1/PD-L1/PD-L2 axis.
In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [A Group, Type 3] Su strain.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes. 



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1: Evaluation of EMT6 triple negative breast cancer tumor growth (pre-randomization) in the 54 mice enrolled in the study.
FIG. 2: In vivo efficacy results with an exemplary composition comprising non-viable cells of Streptococcus pyogenes (“Composition 002” or “Comp. 002”) in the EMT6 Model.
FIGS. 3A-3B: In vivo efficacy results with intratumor (FIG. 3A) or intravenous (FIG. 3B) delivery of Composition 002 as a single agent and in combination with anti-PD1 antibody.
FIGS. 4A-4B: Body weigh measure data of primary tumors (FIG. 4A) and spleens (FIG. 4B) of mice dosed with Composition 002 intratumorally as a single agent and in combination with anti-PD1 antibody.
FIGS. 5A-5B: Weigh measure data of primary tumors (FIG. 5A) and spleens (FIG. 5B) of mice dosed with Composition 002 intravenously as a single agent and in combination with anti-PD1 antibody.
FIGS. 6A-6B: Measure of mouse body weights average (FIG. 6A) and percent body weight change (FIG. 6B) in Balb/c mice bearing EMT6 tumors (pre-randomization).
FIGS. 7A-7B: Measure of mouse body weights average (FIG. 7A) and percent body weight change (FIG. 7B) in Balb/c mice bearing EMT6 tumors (post-randomization, all groups).
FIGS. 8A-8B: Measure of percent body weight change (post-randomization) in Balb/c mice bearing EMT6 tumors in the intratumor delivery groups (FIG. 8A) and intravenous delivery groups (FIG. 8B).
FIGS. 9A-9B: Analysis of % CD3+ T cells (gated on CD45+ CD3+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 9A) and intratumor groups (FIG. 9B).
FIGS. 10A-10B: Analysis of CD4+ T cells (gated on CD45+ CD3+ CD8− CD4+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 10A) and intratumor groups (FIG. 10B).
FIGS. 11A-11B: Analysis of CD8+ T cells (gated on CD45+ CD3+ CD4− CD8+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 11A) and intratumor groups (FIG. 11B).
FIGS. 12A-12B: Analysis of NK+ cells (gated on CD45+ CD3− CD49b+-CD335+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 12A) and intratumor groups (FIG. 12B).
FIGS. 13A-13B: Analysis of percent granulocytes MDSCs cells (gated on CD45+ CD3− CD11+−b-Ly6G+Ly6Clow) and Monocytes MDSCs in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 13A) and intratumor groups (FIG. 13B).
FIGS. 14A-14B: Analysis of the ratio granulocytes MDSCs cells (gated on CD45+ CD3− CD11b+−Ly6G+Ly6Clow) vs. Monocytes MDSCs in spleen (Left) and tumors-TTLs (Right) of the intravenous treatment groups (FIG. 14A) and intratumor groups (FIG. 14B).
FIGS. 15A-15B: Analysis of regulatory T cells (gated on CD45+ CD3+CD4+ CD25+Fox3+) in spleen (Left) and tumors-TTLs (Right) of the intravenous treatment groups (FIG. 15A) and intratumor groups (FIG. 15B).
FIGS. 16A-16B: Analysis of TAMs (M1/M2 ratio): gated on CD45+ CD3− F4/80+ CD206− (M1) or CD45+ CD3− F4/80+ CD206+ (M2) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 16A) and intratumor groups (FIG. 16B).
FIGS. 17A-17B: Analysis of PD1+/high T cells (gated on CD45+ CD3+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 17A) and intratumor groups (FIG. 17B).
FIGS. 18A-18B: Analysis of PD1+/high macrophages (gated on CD45+ CD3− F4/80+)+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 18A) and intratumor groups (FIG. 18B).
FIGS. 19A-19B: Analysis of PD-L1+/high T cells (gated on CD45+ CD3+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 19A) and intratumor groups (FIG. 19B).
FIGS. 20A-20B: Analysis of PD-L1+/high macrophages (gated on CD45+ CD3− F4/80+) in spleen (Left) and tumors-TILs (Right) of the intravenous treatment groups (FIG. 20A) and intratumor groups (FIG. 20B).
FIGS. 21A-21K: Summary of the flow cytometry analysis data for Composition 002 (dose 20 mg/Kg) in the splenocytes and TILs of the intratumor delivery groups: CD3+ T cells (FIG. 21A), CD4+ T cells (FIG. 21B), CD8+ T cells (FIG. 21C), NK cells (FIG. 21D), MDSCs/Monocytes ratio (FIG. 21E), Treg cells (FIG. 21F), Macrophages M1/M2 ratio (FIG. 21G), PD1+ T cells (FIG. 21H), PD-L1+ T cells (FIG. 21I), PD-L1+ macrophages (FIG. 21J), and PD-L1+ macrophages (FIG. 21K).
FIGS. 22A-22B: Antitumor Efficacy of Composition 002 and Anti-mPD-1 in Mono- and Combination Therapy in the MBT-2 Tumor Model. (FIG. 22A) Mean absolute tumor volumes±SEM over time; (FIG. 22B) Individual absolute tumor volumes on Day 7 (the final day on which all groups remained on study).
FIGS. 23A-23F: Effect of Composition 002 and Anti-mPD-1 Treatment on Leukocyte Populations in MBT-2 Tumors Implanted subcutaneously (s.c.) in C3H Mice—Cell Percentages. FC analysis of cells isolated from the MBT-2 tumors from 10 mice per group at the end timepoint on Day 10 (Groups 1 and 8) or Day 8 (Groups 2-7). Cells were stained with the antibody panels A and B as described in Example 2. The X-axis indicates the population of interest which is presented for each individual animal as the percentage of the parent population indicated in the red text underneath the X-axis label. The horizontal bar in each data set indicates the group mean value. (FIG. 23A) Antibody panel A: CD45+ cells; (FIG. 23B) Antibody panel A: CD4+/CD8+ T cells and Treg cells; (FIG. 23C) Antibody panel A: granulocytic and monocytic MDSC; (FIG. 23D) Antibody panel B: CD45+ CD3− CD11b+ cells; (FIG. 23E) Antibody panel B: NK cells; (FIG. 23F) Antibody panel B: M1 and M2 macrophages.
FIGS. 24A-24F: Effect of Composition 002 and Anti-mPD-1 Treatment on Leukocyte Populations in MBT-2 Tumors Implanted subcutaneously (s.c.) in C3H Mice—Cell Counts. FC analysis of cells isolated from the MBT-2 tumors from 10 mice per group at the end timepoint on Day 10 (Groups 1 and 8) or Day 8 (Groups 2-7). Cells were stained with the antibody panels A and B given under as described in Example 2. The X-axis indicates the population of interest which is presented for each individual animal as the cell counts within the population indicated in the red text below the X-axis label. The horizontal bar in each data set indicates the group mean value. (FIG. 24A) Antibody panel A: CD45+ cells; (FIG. 24B) Antibody panel A: CD4+/CD8+ T cells and Treg cells; (FIG. 24C) Antibody panel A: granulocytic and monocytic MDSC; (FIG. 24D) Antibody panel B: CD45+ CD3− CD11b+ cells; (FIG. 24E) Antibody panel B: NK cells; (FIG. 24F) Antibody panel B: M1 and M2 macrophages.
FIG. 25: Impact of Treatment on Body Weights of Mice. The group mean relative body weights over time of all groups are shown.
FIG. 26: Composition 002 treatment does not change the number of CD4+ and CD8+ T cells.
FIGS. 27A-27B: Expression of Immune Checkpoint Molecules in Composition 002 treated (FIG. 27A) CD4+ T cells and (FIG. 27B) CD8+ T cells (average of two donors). Data are expressed as mean (two donors/triplicate)±SD. **=P<0.01. * * *=P<0.001.
FIGS. 28A-28D: COMPOSITION-002 treatment leads to tumor apoptosis and release of Damage-associated molecular pattern molecules (DAMPs). MB49 cells were treated with different concentrations of COMPOSITION-002 for 24 hrs. Mitoxantrone (1 uM) was used as positive control. FIG. 28A: Percentage of Annexin V positive MB49 cells were measured by Flow Cytometry as a marker for apoptosis. Data is presented as Mean±SEM. FIG. 28B: Calreticulin-positive MB49 cells quantified by Flow cytometry. FIG. 28C. Extracellular ATP (eATP) luminescence was measured and quantified using the formula: [(average of triplicate cellstreated RLU−average of cell−free media+drug RLU)/(average of cellsuntreated RLU−cell−free media RLU)×100−100]. Where RLU represents background-subtracted luminescence. FIG. 28D. After 24 hours of treatment, supernatant was harvested and HMGB1 was quantified using Lumit Immunoassay. FIGS. 28B-28D Data is presented Average Fold Change±SEM over the untreated control group. One-way ANOVA with Tuckey's post test *, P<0.05; **, P<0.01; ****, P<0.0001. N=3.
FIGS. 29A-29D: MB49 cells previously exposed to COMPOSITION-002, leads to Dendritic Cells (DCs) maturation and higher phagocytosis rate. MB49 cells were previously treated with different concentrations of COMPOSITION-002 for 24 hrs. The drug was removed, and the tumor cells were co-cultured for an additional 24 hrs with DCs isolated from C57bl/6 mouse bone marrow. CD86-positive (FIG. 29A), CD80-positive (FIG. 29B) and HLA-DR-positive (FIG. 29C) Dendritic cells (CD11+) were quantified by Flow Cytometry as markers of cell maturation. FIG. 29D: To evaluate phagocytosis, co-localization of pre-labeled tumor cell signal (DiO+) with DC cells (CD11+) were quantified by Flow Cytometry. Dinaciclib (1 uM) was used as positive control. Data is presented as Mean±SEM. One-way ANOVA with Tuckey's post test *, P<0.05; **; P<0.01; ***, P<0.001; ****, P<0.0001. N=3.
FIGS. 30A-30B: COMPOSITION-002 was effective in promoting immune mediated killing of tumor cells. Untreated cells (left bar); COMPOSITION-002 treated cells (right bar). FIG. 30A: Human bladder cancer cells 5637 and RT112 were treated with COMPOSITION-002 (0.2 KE/mL) for 72 hs. FIG. 30B: Human bladder cancer cells 5637 and RT112 were co-cultured with PBMCs with or without COMPOSITION-002 (0.2 KE/mL) for 72 hs. Tumor cell viability was accessed by Flow Cytometry using Live/Dead dye. Data is presented as Mean±SEM. T-test; **; P<0.01; * * * *, P<0.0001. N=3; One PBMC donor was used.
FIGS. 31A-31B: COMPOSITION-002 treatment induced Th1 and reduced Th2 cytokines release. Human bladder cancer cells 5637 and RT112 were co-cultured with PBMCs and COMPOSITION-002 (0.2 KE/mL) for 72 hs. Supernatant was collected, and a panel of pro-inflammatory cytokines were measured by electrochemiluminescence detection (MSD assay). Untreated cells (left bar); COMPOSITION-002 treated cells (right bar). FIG. 31A: Th1 cytokines (from left to right: IFN-γ, TNF-α and IL-12p70) and FIG. 31B: Th2 cytokines (from left to right: IL-13, IL-10, and IL-4) were quantified. Data is presented as Mean±SEM. T-test; *, P<0.05; **; P<0.01; * * *, P<0.001; * * * *, P<0.0001. N=3; One PBMC donor was used.
FIGS. 32A-32M: COMPOSITION-002 induced T cell proliferation and activation, however it also increased markers of exhausted phenotype. T cells isolated from PBMC were treated with COMPOSITION-002 (0.2 KE/mL) for 72 hs. CD4+ T cells were analyzed for the proliferation marker Ki67 (FIG. 32A), as well as the LAG3 (FIG. 32B), CTLA4 (FIG. 32C), PD-1 (FIG. 32D), TIGIT (FIG. 32E), TIM3 (FIG. 32F) and FOXP3 (FIG. 32G) markers. FIG. 32H: CD8 T cell proliferation was assessed by quantification of Ki67-positive cells by flow cytometry. CD8+ T cells' CTLA4 (FIG. 32I), LAG3 (FIG. 32J), PD-1 (FIG. 32K), TIGIT (FIG. 32L) and TIM3 (FIG. 32M) markers were also analyzed by Flow cytometry. Data is presented as Mean±SEM of 2 different donors in triplicate. N=6. T-test; **; P<0.01; ***, P<0.001.
FIGS. 33A-33B: COMPOSITION-002 activates T cells by increasing IFN-7 and Granzyme B release. T cells isolated from PBMC were treated with COMPOSITION-002 (0.2 KE/mL) for 72 hs. FIG. 33A: IFN-7 and FIG. 33B: Granzyme B were detected and quantified by ELISA assay. Data is presented as Mean±SEM of 2 different donors in triplicate. N=6. T-test; * *; P<0.01; * **, P<0.001.
FIG. 34: COMPOSITION-002 treatment induces PD-L1 expression in 5637 bladder tumor cells in co-culture. T cells isolated from PBMC were co-cultured with the human bladder cancer cell 5637 and COMPOSITION-002 (0.2 and 0.8 KE/mL) for 72 hs. PD-L1 signal was quantified in pre-labeled 5637 by Flow Cytometry. Data are presented as Mean±SEM. N=3. T-test; *, P<0.05; **; P<0.01; * * *, P<0.001.
FIGS. 35A-35F: Anti-PD-L1 and anti-CTLA4 synergize with COMPOSITION-002. In vitro cytotoxicity was measured using xCELLigence real-time cell analyzer. Human bladder cancer 5637 cells were seeded in 96-well E-Plate and allow to grow for 78 h. Subsequently, the effector cells (human PBMCs; effector/target ratio 6.6:1) and treatments were added. COMPOSITION-002 treatment (0.8 KE/mL) was carried out for −65 hours in the co-culture setting (PBMC+ Tumor cells) alone or in combination with the following antibodies: (FIGS. 35A-35B) anti-PD-L1, (FIGS. 35C-35D) anti-CTLA-4, (FIGS. 35E-35F) anti-PD-1. Irrelevant IgG4, and IgG1 were used as isotype controls. Impedance measurements were collected by xCelligence RTCA eSight (Agilent) every 15 min. Same data as from (FIGS. 35A, 35C, and 35E) but compared with each group at one specific time point (143 h:49 m:32 s) shown as bar charts (FIGS. 35B, 35D, and 35F, respectively) with error bars. Data are presented as Mean SEM. N=4. Statistical significance was determined using the two-way ANOVA and Bonferroni Post test *, P<0.05; **, P<0.01; ***P<0.001; * * * *, P<0.0001 (n=4).
FIGS. 36A-36B: COMPOSITION-002 treatment reduces tumor growth and expands animal survival in the anti-PD-1 sensitive MB49 subcutaneous mouse model. MB49 bladder cancer cells were implanted subcutaneously in C57bl/6 mice. After 7 days, animals were randomized and received COMPOSITION-002 treatment (2, 0.4 or 0.8 KE/mouse) once a week intravenously for 4 weeks. FIG. 36A: Tumor Volume was analyzed biweekly using calipers. Data is presented as Mean Tumor Volume. N=10, Two-way ANOVA and Bonferroni Post test; **; P<0.01 compared to the untreated control (Saline Vehicle). FIG. 36B: Kaplan Meier curve comparing percentage of survival rates between COMPOSITION-002 treated animals and the Vehicle control. N=10.
FIGS. 37A-37E: COMPOSITION-002 in combination with the checkpoint inhibitor Anti-PD-1 led to reduced tumor size in the MB49 subcutaneous mouse model. MB49 cells were implanted subcutaneously in C57bl/6 mice. After 6 days, animals were randomized and received COMPOSITION-002 treatment (0.4 KE/mouse) once a week intravenously for 4 weeks and Anti-PD-1 treatment (10 mg/Kg) intraperitoneally twice a week for 2 weeks. FIG. 37A: Tumor Volume was analyzed biweekly using calipers. Data is presented as Mean±SEM. N=10, Two-way ANOVA and Bonferroni Post test; **; P<0.01; * * * *, P<0.0001 compared to the untreated control (Isotype Control). FIG. 37B: Detailed Tumor Volume curve comparing Anti-PD-1 as a monotherapy and COMPOSITION-002+Anti-PD-1 in combination. Data is presented as Mean±SEM. N=10. FIG. 37C: Histogram analysis of average tumor volume from mice treated with Anti-PD-1 as a monotherapy or COMPOSITION-002+Anti-PD-1 in combination at Day 20 of treatment. FIG. 37D: Tumor Growth progression in mice treated with Anti-PD-1. Light green lines indicate individual mice enrolled in the study; whole dark green line represents the average tumor volume. FIG. 37E: Tumor Growth progression in mice treated with Anti-PD-1. Light purple lines indicate individual mice enrolled in the study whole dark purple line represents the average tumor volume.
FIGS. 38A-38B: COMPOSITION-002 treatment reduces tumor growth in in PD-1 resistant triple negative breast cancer model associated with increase expression of PD-1 in T cells. The Anti-PD-1 resistant triple negative breast cancer cells (EMT6) were implanted orthotopically in female Balb/c mice. After 8 days, mice were randomized and received COMPOSITION-002 (0.4, 1 and 2 KE/mouse) treatment twice a week intravenous for 3 weeks.
FIG. 38A: Tumor volume was measured using calipers. Data is presented as Mean±SEM. N=6, T-test; *; P<0.05. FIG. 38B: On day 22 after treatment initiation, mice were euthanized, tumors were collected, processed stained for Flow Cytometry analysis of tumor infiltrated immune cells. Tumor infiltrated T cells were analyzed for presence of PD-1 marker (left to right: control; COMP-002 0.4 KE/mouse; COMP-002 1 KE/mouse; COMP-002 2 KE/mouse). Data presented as Mean±SEM. One-Way ANOVA Dunnett's Post Test. **, P<0.01, ***, P<0.001. N=3.
FIGS. 39A-39B: COMPOSITION-002 in combination with Anti-PD-1 revealed reduced tumor volume and weight in the anti-PD-1 resistant EMT6 orthotopic mouse model. The anti-PD-1 resistant triple negative breast cancer cells were implanted orthotopically in female Balb/c mice. After 8 days, mice were randomized and received COMPOSITION-002 (10 mg/Kg) treatment twice a week intravenously for 3 weeks and Anti-PD-1 treatment (100 ug/mouse) intraperitoneally twice a week for 2 weeks. FIG. 39A: Tumor Volume was measured biweekly using calipers. Data is presented as Mean±SEM. N=6, One-way ANOVA and Bonferroni Post test; *; P<0.05; **, P<0.001 compared to the untreated control (Saline Control). FIG. 39B: Tumor weight was quantified at the end of the study (Day 30 after tumor implantation). Data is presented as Mean±SEM. One-Way ANOVA Tucket Post test. **, P<0.001. N=6.
FIGS. 40A-40D: COMPOSITION-002 and Anti-PD-1 combination therapy in EMT6 orthotopic mouse model reshaped the tumor microenvironment. The Anti-PD-1 resistant triple negative breast cancer cells were implanted orthotopically in female Balb/c mice. After 8 days, mice were randomized and received COMPOSITION-002 treatment (10 mg/Kg) twice a week intravenously for 3 weeks and Anti-PD-1 treatment (200 ug/Kg) intraperitoneally twice a week for 2 weeks. On day 30 post tumor implantation, mice were euthanized, and primary tumors were collected, processed, and stained for Flow Cytometry analysis of tumor infiltrated immune cells. Tumor Infiltrated analysis of T cells and PD-1-positive T cells (FIG. 40A); macrophage cells subpopulations (FIG. 40B); C. T regulatory cells (FIG. 40C); Natural Killer (NK) cells (FIG. 40D). Data shown from left to right: Control, Anti-PD-1, COMPOSITION-002, and COMPOSITION-002+anti-PD-1. Data presented as Mean±SEM. One-Way ANOVA Tukey's post test. *, P<0.05, **, P<0.01, ***, P<0.001; ****, P<0.0001. N=3.



DETAILED DESCRIPTION
Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer) or subranges, unless otherwise indicated.
As used herein, the term “about” means+20% of the indicated range, value, or structure, unless otherwise indicated.
It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.
As used herein, the terms “include,” “have,” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
“Optional” or “optionally” means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.
The term “antibody” refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the 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, nanobody). The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
As used herein, the term “immune checkpoint molecule” refers to one or more proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response. For example, immune checkpoint molecules include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression. “Controlling or suppressing an immune response,” as used herein, 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 modulation, control or suppression can promote or permit the persistence of a hyperproliferative disease or disorder (e.g., cancer, chronic infections).
Exemplary immune checkpoint molecules include immune checkpoint ligands (such as PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9), immune checkpoint receptors (such as PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR), metabolic enzymes (such as arginase, indoleamine 2,3-dioxygenase (IDO)), immunosuppressive cytokines (such as IL-10, IL-4, IL-1RA, IL-35), Treg cells, or any combination thereof. In certain embodiments, an immune checkpoint molecule may initiate an immune suppression signal through a ligand-receptor interaction, such as by modulating (e.g., inhibiting) an antigen-specific T cell response. For example, a T cell may express on its surface an immune checkpoint receptor (e.g., PD-1, LAG3) and an antigen presenting cell may express on its surface an immune checkpoint receptor ligand (e.g., PD-L1, MHC/HLA molecule). In further embodiments, an immune checkpoint molecule is a metabolic enzyme that inhibits immune responses through the local depletion of amino acids essential for lymphocyte, particularly T cell, survival and function. In still further embodiments, an immune checkpoint molecule may be a signaling molecule, such as an immunosuppressive cytokine (e.g., IL-10, IL-4, IL-1RA, IL-35).
Furthermore, an immune checkpoint molecule (e.g., IL-10) may cause a reduction in the expression or level of a major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecule, which can in turn reduce antigen presentation and thereby reduce, impede or detectably prevent T cell activation and a corresponding immune response.
An “immune checkpoint inhibitor” refers to any molecule that can alter, interfere, reduce, downregulate, block, suppress, abrogate, degrade, directly or indirectly, expression, amount, or activity of an immune checkpoint molecule. Exemplary immune checkpoint inhibitors include small molecules, nucleic acid molecules (including vaccines, such as mRNA vaccines, and inhibitory nucleic acids such as antisense oligonucleotides, siRNAs, shRNAs, and miRNAs), peptides, proteins, antibodies or antigen binding fragments thereof, fusion proteins, ribozymes, or gene editing systems.
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.
As used herein, “non-muscle invasive bladder cancer” or “NMIBC” refers to urothelial carcinoma confined to the bladder mucosa and submucosa and does not invade into or beyond the muscularis propia.
As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition of the present disclosure, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of an animal model.
As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition of the present disclosure, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of an animal model.
Composition Comprising Non-Viable Cells of Streptococcus pyogenes 
The present disclosures provides a composition comprising non-viable cells of Streptococcus pyogenes and an immune checkpoint inhibitor for use in combination with an immune checkpoint inhibitor to treat a subject with triple negative breast cancer or non-muscle invasive bladder cancer.
Streptococci are Gram-positive, catalase-negative, coagulase-negative cocci that occur in pairs or chains. They are divided into three groups by the type of hemolysis on blood agar: beta-hemolytic (complete lysis of red cells), a hemolytic (green hemolysis), and gamma-hemolytic (no hemolysis). Beta-hemolytic streptococci are characterized as group A streptococci (Streptococcus pyogenes) and group B streptococci (Streptococcus agalactiae). Streptococcus pyogenes are Gram-positive, non-spore forming coccus-shaped bacteria which typically exist in chains or in pairs of cells. S. pyogenes are subdivided according to serotype based on a large, highly variable cell surface antigen call the M protein (Lancefield, J. Exp. Med. 47, 9-10, 1928; Lancefield, J. Immunol. 89, 307-13, 1962). DNA sequencing of genes encoding M proteins has become the most common method of determining S. pyogenes M types (emm sequence types). To date, 124 different M types have been identified (Facklam et al., Clin. Infect. Dis. 34, 28-38, 2002). M1, M28, M12, M3, M11, and M6 are among the most prevalent GAS types worldwide (Li et al., Infect. Dis. 188, 1587-92, 2003; O'Brien et al., Clin. Infect. Dis. 35, 268-76, 2002). Any strain of Streptococcus pyogenes (S. pyogenes) is envisaged for use within the disclosure of the instant claims. In some embodiments of the disclosure, the strain of S. pyogenes used is selected by the strain M protein (serotype). In some embodiments, the strain is an invasive strain. In some embodiments, the strain was isolated from a clinical sample. In some embodiments, the strain is a 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.
In some embodiments, the composition comprises Streptococcus pyogenes M protein type 3.
In some embodiments, the composition comprises Streptococcus pyogenes (A Group, Type 3) Su strain.
In some embodiments of the disclosure of the instant claims, the strain of S. pyogenes used is selected from the group consisting of the strains that have been deposited with the American Type Culture Collection (ATCC). In some embodiments, the instant disclosure provides for a mixture comprising more than one strain of S. pyogenes. In some embodiments, the mixture comprises the Su strain and at least one additional strain of S. pyogenes. Exemplary S. pyogenes strains, including the Su strain, that are envisaged for use in the present disclosure are described in Table A. Additional information on individual strains is described at: https://www.atcc.org/search #q=streptococcus%20pyogenes&sort=relevancy&numberOfResults=24&f:Productcategory=[Bacteria], which is incorporated herein by reference. In some embodiments, the composition comprises any one or more of the S. pyogenes strains identified in Table A below:







TABLE A







Exemplary strains of S. pyogenes









ATCC 


Strain designation
Number











Su
21060


Bruno [CIP 104226]
19615


Typing strain T1 [NCIB 11841, SF 130]
12344


SF370; M1 GAS
700294


Richards-L
19563


QC A62
49399


C203 S
14289


MGAS 6180 [BE98-762]
BAA-1064


MGAS 10394
BAA-946


MGAS 315
BAA-595


NCTC 8709 (Type 6 glossy)
12203


NCTC 8370 (Type I)
12202


Typing strain C203 [Dochez 1708]
12384


NZ131
BAA-1633


MGAS 10270
BAA-1063


MGAS 5005
BAA-947


Blackmore
21548


D58 [ATCC 12346, ATCC 9959]
10389


397
49117


1805 [ON1763]
51339


Typing strain T14
12972


NCTC 8306 [Mathews type 25]
12204


Typing strain C94 [13RS1]
12370


H3
11434


[40]
624


MGAS 2096 [A374]
BAA-1065


Typing strain C121 [19RS63]
12364


Typing strain J17C [A. Coburn R20]
12357


MGAS 10750 [FL01-86]
BAA-1066


MGAS 8232
BAA-572


MGAS9429 Serotype M12
BAA-1315


S-43
21547


C 203 S
21546


Typing strain T5B [F. Griffith strain Franklin]
12347


Typing strain C265 [F. Griffith NF14 or SFH]
12349


Typing strain T22 [F. Griffith 63T]
10403


Typing strain T23 [F. Griffith strain Barts 102]
8133


[Kjem's 3807, NIH 61 x 99]
14918


[Kjem's strain K56, NIH 61 x 101]
14919


[P20080]
25663


SH
8058


Wilders D58
43202


NYDP 14E [ATCC 6550, Dochez 5]
4543


S376
51877


12151
51878


M-3 [DLS 88002, Weller]
51500


22
51574


CDC-SS-1435 [MstNS1]
BAA-361


CDC-SS-1437 [MstNS14X]
BAA-363


CDC-SS-1462 [MstNS5]
BAA-362


CDC-SS-1433 [Mst90/85]
BAA-360


CDC-SS-1434 [Mst88/25]
BAA-359


CDC-SS-1475 [Mst13w, R90/865]
BAA-355


CDC-SS-1432 [Mst88/31]
BAA-358


CDC-SS-1343 [Mst64/14]
BAA-356


CDC-SS-1413 [MstA207]
BAA-357


59388
BAA-1414


Typing strain T28
12962


Typing strain B403
12963


Typing strain C649A
12961


Typing strain C510
12964


Typing strain C113 [RS79]
12374


Typing strain B447 [R. Williams strain Corby B6522]
12381


Grouping strain J17A4 [A.F. Coburn RPHI]
12385


Typing strain B514 [J. Nelson 7353]
12382


London [E. Todd strain London]
12379


Typing strain H105op [W. Tillett strain CO]
12380


Typing strain C126 [2RS63]
12375


Typing strain D58X
12383


Typing strain C744 [F. Griffith SF 13]
12378


C105 [20RS14]
12377


Typing strain C143 [C143]
12372


Typing strain C101 [4RS8]
12373


Typing strain C142 [A. Kuttner B22]
12366


Typing strain C95 [19RS14]
12371


Typing strain J137
12363


Typing strain C119 [A. Kuttner B35]
12368


Typing strain C171 [A. Kuttner 24 Berg]
12367


Typing strain D24/94/1 [F. Griffith strain Quinn]
12362


Typing strain C107 [14RS60]
12365


Typing strain B346op [41459]
12359


Typing strain J17E [A. Coburn R9]
12356


Typing strain T13 [ATCC 6553, F. Griffith strain
12354


Glover]



Start page 3



Typing strain C95 [19RS14]
12371


Typing strain J17F [A. Coburn R17]
12360


Typing strain C119 [A. Kuttner B35]
12368


Typing strain C171 [A. Kuttner 24 Berg]
12367


Typing strain D24/94/1 [F. Griffith strain Quinn]
12362


Typing strain C107 [14RS60]
12365


Typing strain B346op [41459]
12359


Typing strain J17E [A. Coburn R9]
12356


Typing strain T13 [ATCC 6553, F. Griffith strain
12354


Glover]



Typing strain J17F [A. Coburn R17]
12360


Typing strain E14 [Dochez NY 5]
12351


Typing strain T12 [F. Griffith SF 42]
12353


Typing strain J17D [A. Coburn R3]
12358


Typing strain T11 [F. Griffith NE 73T]
12352


Typing strain S43 [Dochez and Avery S43 (Texas)]
12348


Typing strain T9 [F. Griffith strain Symons]
12350


Typing strain T2 [BIT]
12345


C115 [G54]
10781


Typing strain C98
10782


Typing strain Coggins D23
10096


35
11435


8
11436


Typing strain T27 [780 Tate]
8135


C203G
8669


C203M [NCIB 8884, PCI 1307]
8668


C203R
8670


1685M
8671


[77/4523]
29218


Typing strain T15 [F. Griffith JS 5]
9898


CDC-SS-1173 [1233]
700503


CDC-SS-1152 [R75/2681]
700502


CDC-SS-1151 [R74/2015]
700501


CDC-SS-1150 [R72/3085]
700500


CDC-SS-1098 [Cairo 5]
700493


CDC-SS-1146 [R67/1720]
700496


CDC-SS-1097 [Cairo 4]
700490


CDC-SS-1144 [R65/3961]
700494


CDC-SS-1149 [PT2773, R72/2773]
700499


CDC-SS-1147 [R68/3354]
700497


CDC-SS-1042 [5654-15]
700485


CDC-SS-1145 [R67/239]
700495


CDC-SS-989 [644]
700484


CDC-SS-984 [R68/485]
700482


CDC-SS-1148 [PT Furo, R72/943]
700498


CDC-SS-1096 [Cairo 3]
700489


CDC-SS-985 [SF2]
700483


CDC-SS-875 [2998-T, Alabama 11]
700481


CDC-SS-1037 [M66 vaccine strain]
700486


CDC-SS-1399 [NCTC 12065, PT-2841, R76/2841]
700947


CDC-SS-1448 [PT-2631, PT-Trinidad 2631,
700946


R76/2631]



CDC-SS-1402 [NCTC 12062, PT-180, R72/180,
700942


SS-1395]



CDC-SS-1400 [NCTC 12064, PT-Leeds 2110,
700943


R82/2110]



CDC-SS-1396 [NCTC 12068, PT-4931, R61/2516]
700950


CDC-SS-1398 [NCTC 12066, PT-5757, R79/5757]
700951


CDC-SS-1493 [Potter 41, R80/5991]
700953


CDC-SS-1460 [2974-95, PT-NZ5118, SF1617A]
700952


59221
BAA-1411


59650
BAA-1413


56889
BAA-1415


65512
BAA-1412


Typing strain T27 [780 Tate]
8135


C203G
8669


C203M [NCIB 8884, PCI 1307]
8668


C203R
8670


1685M
8671


[77/4523]
29218


YL15
BAA-1324


YL16
BAA-1325


YL17
BAA-1326


ALAB49
BAA-1323


Typing strain T15 [F. Griffith JS 5]
9898


B737 [Wanamaker strain Red Lake 12 Sp. II]
13540


C203 U
27762


P20080-L
27080









In some embodiments, the composition comprising non-viable Streptococcus pyogenes is a pharmaceutical composition and optionally comprises at least one pharmaceutically acceptable excipient, for example, a stabilizing agent, a buffering agent, a bulking agent, an antioxidant, a tonicity agent, an antimicrobial agent, or any combination thereof. As is known to one skilled in the art, in some instances a component may 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) may function as both a bulking agent and a tonicity agent.
In some embodiments, the composition is a lyophilized composition.
In some embodiments, the stabilization agent is selected from magnesium hydroxide, calcium hydroxide, calcium carbonate, magnesium oxide, magnesium carbonate, magnesium sulfate, and a combination thereof.
In some embodiments, the stabilization agent is magnesium hydroxide. In some embodiments, the stabilization agent is calcium hydroxide. In some embodiments, the stabilization agent is calcium carbonate. In some embodiments, the stabilization agent is magnesium oxide. In some embodiments, the stabilization agent is magnesium carbonate. In some embodiments, the stabilization agent is magnesium sulfate.
In some embodiments, the stabilization agent is present in the lyophilized composition from about 0.10% (w/w) to about 10.00% (w/w), from about 0.10% (w/w) to about 5.00% (w/w), from about 0.10% (w/w) to about 4.50% (w/w), from about 0.10% (w/w) to about 4.00% (w/w), from about 0.10% (w/w) to about 3.50% (w/w), from about 0.10% (w/w) to about 3.00% (w/w), from about 0.10% (w/w) to about 2.50% (w/w), from about 0.10% (w/w) to about 2.00% (w/w), from about 0.10% (w/w) to about 1.50% (w/w), from about 0.10% (w/w) to about 1.00% (w/w), or from about 0.10% (w/w) to about 0.50% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 0.50% (w/w) to about 10.00% (w/w), from about 0.50% (w/w) to about 5.00% (w/w), from about 0.50% (w/w) to about 4.50% (w/w), from about 0.50% (w/w) to about 4.00% (w/w), from about 0.50% (w/w) to about 3.50% (w/w), from about 0.50% (w/w) to about 3.00% (w/w), from about 0.50% (w/w) to about 2.50% (w/w), from about 0.50% (w/w) to about 2.00% (w/w), from about 0.50% (w/w) to about 1.50% (w/w), or from about 0.50% (w/w) to about 1.00% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 1.00% (w/w) to about 10.00% (w/w), from about 1.00% (w/w) to about 5.00% (w/w), from about 1.00% (w/w) to about 4.50% (w/w), from about 1.00% (w/w) to about 4.00% (w/w), from about 1.00% (w/w) to about 3.50% (w/w), from about 1.00% (w/w) to about 3.00% (w/w), from about 1.00% (w/w) to about 2.50% (w/w), from about 1.00% (w/w) to about 2.00% (w/w), or from about 1.00% (w/w) to about 1.50% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 1.50% (w/w) to about 10.00% (w/w), from about 1.50% (w/w) to about 5.00% (w/w), from about 1.50% (w/w) to about 4.50% (w/w), from about 1.50% (w/w) to about 4.00% (w/w), from about 1.50% (w/w) to about 3.50% (w/w), from about 1.50% (w/w) to about 3.00% (w/w), from about 1.50% (w/w) to about 2.50% (w/w), or from about 1.50% (w/w) to about 2.00% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 2.00% (w/w) to about 10.00% (w/w), from about 2.00% (w/w) to about 5.00% (w/w), from about 2.00% (w/w) to about 4.50% (w/w), from about 2.00% (w/w) to about 4.00% (w/w), from about 2.00% (w/w) to about 3.50% (w/w), from about 2.00% (w/w) to about 3.00% (w/w), or from about 2.00% (w/w) to about 2.50% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 2.50% (w/w) to about 10.00% (w/w), from about 2.50% (w/w) to about 5.00% (w/w), from about 2.50% (w/w) to about 4.50% (w/w), from about 2.50% (w/w) to about 4.00% (w/w), from about 2.50% (w/w) to about 3.50% (w/w), or from about 2.50% (w/w) to about 3.00% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 3.00% (w/w) to about 10.00% (w/w), from about 3.00% (w/w) to about 5.00% (w/w), from about 3.00% (w/w) to about 4.50% (w/w), from about 3.00% (w/w) to about 4.00% (w/w), or from about 3.00% (w/w) to about 3.50% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 3.50% (w/w) to about 10.00% (w/w), from about 3.50% (w/w) to about 5.00% (w/w), from about 3.50% (w/w) to about 4.50% (w/w), or from about 3.50% (w/w) to about 4.00% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 4.00% (w/w) to about 10.00% (w/w), from about 4.00% (w/w) to about 5.00% (w/w), or from about 4.00% (w/w) to about 4.50% (w/w).
In some embodiments, the stabilization agent is present in the lyophilized composition from about 4.50% (w/w) to about 10.00% (w/w) or from about 4.50% (w/w) to about 5.00% (w/w).
In some embodiments, the buffering agent is a phosphate salt. In some embodiments, the buffering agent is selected from potassium dihydrogen phosphate, sodium phosphate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen phosphate, and sodium dihydrogen phosphate and a combination thereof.
In some embodiments, the buffering agent is potassium dihydrogen phosphate. In some embodiments, the buffering agent is sodium phosphate.
In some embodiments, the buffering agent is present in the lyophilized composition from about 1.00% (w/w) to about 25.00% (w/w), from about 1.00% (w/w) to about 24.00% (w/w), from about 1.00% (w/w) to about 23.00% (w/w), from about 1.00% (w/w) to about 22.00% (w/w), from about 1.00% (w/w) to about 21.00% (w/w), from about 1.00% (w/w) to about 20.00% (w/w), from about 1.00% (w/w) to about 19.00% (w/w), from about 1.00% (w/w) to about 18.00% (w/w), from about 1.00% (w/w) to about 17.00% (w/w), from about 1.00% (w/w) to about 16.00% (w/w), from about 1.00% (w/w) to about 15.00% (w/w), from about 1.00% (w/w) to about 14.00% (w/w), from about 1.00% (w/w) to about 13.00% (w/w), from about 1.00% (w/w) to about 12.00% (w/w), from about 1.00% (w/w) to about 11.00% (w/w), from about 1.00% (w/w) to about 10.00% (w/w), from about 1.00% (w/w) to about 9.00% (w/w), from about 1.00% (w/w) to about 8.00% (w/w), from about 1.00% (w/w) to about 7.00% (w/w), from about 1.00% (w/w) to about 6.00% (w/w), from about from about 1.00% (w/w) to about 5.00% (w/w), from about 1.00% (w/w) to about 4.00% (w/w), from about 1.00% (w/w) to about 3.00% (w/w), or from about 1.00% (w/w) to about 2.00% (w/w).
In some embodiments, the buffering agent is present in the lyophilized composition from about 5.00% (w/w) to about 25.00% (w/w), from about 5.00% (w/w) to about 24.00% (w/w), from about 5.00% (w/w) to about 23.00% (w/w), from about 5.00% (w/w) to about 22.00% (w/w), from about 5.00% (w/w) to about 21.00% (w/w), from about 5.00% (w/w) to about 20.00% (w/w), from about 5.00% (w/w) to about 19.00% (w/w), from about 5.00% (w/w) to about 18.00% (w/w), from about 5.00% (w/w) to about 17.00% (w/w), from about 5.00% (w/w) to about 16.00% (w/w), from about 5.00% (w/w) to about 15.00% (w/w), from about 5.00% (w/w) to about 14.00% (w/w), from about 5.00% (w/w) to about 13.00% (w/w), from about 5.00% (w/w) to about 12.00% (w/w), from about 5.00% (w/w) to about 11.00% (w/w), from about 5.00% (w/w) to about 10.00% (w/w), from about 5.00% (w/w) to about 9.00% (w/w), from about 5.00% (w/w) to about 8.00% (w/w), from about 5.00% (w/w) to about 7.00% (w/w), or from 5.00% (w/w) to about 6.00% (w/w).
In some embodiments, the buffering agent is present in the lyophilized composition from about 10.00% (w/w) to about 25.00% (w/w), from about 10.00% (w/w) to about 24.00% (w/w), 1 from about 0.00% (w/w) to about 23.00% (w/w), from about 10.00% (w/w) to about 22.00% (w/w), from about 10.00% (w/w) to about 21.00% (w/w), from about 10.00% (w/w) to about 20.00% (w/w), from about 10.00% (w/w) to about 19.00% (w/w), from about 10.00% (w/w) to about 18.00% (w/w), from about 10.00% (w/w) to about 17.00% (w/w), from about 10.00% (w/w) to about 16.00% (w/w), from about 10.00% (w/w) to about 15.00% (w/w), from about 10.00% (w/w) to about 14.00% (w/w), from about 10.00% (w/w) to about 13.00% (w/w), from about 10.00% (w/w) to about 12.00% (w/w), from about 10.00% (w/w) to about 11.00% (w/w). In some embodiments, the buffering agent is present in the lyophilized composition from about 15.00% (w/w) to about 25.00% (w/w), from about 15.00% (w/w) to about 24.00% (w/w), from about 15.00% (w/w) to about 23.00% (w/w), from about 15.00% (w/w) to about 22.00% (w/w), from about 15.00% (w/w) to about 21.00% (w/w), from about 15.00% (w/w) to about 20.00% (w/w), from about 15.00% (w/w) to about 19.00% (w/w), from about 15.00% (w/w) to about 18.00% (w/w), from about 15.00% (w/w) to about 17.00% (w/w), or from about 15.00% (w/w) to about 16.00% (w/w).
In some embodiments, the buffering agent is present in the lyophilized composition from about 18.00% (w/w) to about 25.00% (w/w), from about 18.00% (w/w) to about 24.00% (w/w), from about 18.00% (w/w) to about 23.00% (w/w), from about 18.00% (w/w) to about 22.00% (w/w), from about 18.00% (w/w) to about 21.00% (w/w), from about 18.00% (w/w) to about 20.00% (w/w), or from about 18.00% (w/w) to about 19.00% (w/w).
In some embodiments, the bulking agent is selected from sodium chloride, mannitol, sucrose, lactose, dextran, trehalose, glycine, maltose and a combination thereof.
In some embodiments, the bulking agent is sodium chloride. In some embodiments, the bulking agent is mannitol. In some embodiments, the bulking agent is sucrose. In some embodiments, the bulking agent is lactose. In some embodiments, the bulking agent is dextran. In some embodiments, the bulking agent is trehalose. In some embodiments, the bulking agent is maltose. In some embodiments, the bulking agent is glycine.
In some embodiments, the bulking agent is present in the lyophilized composition from about 0.05% (w/w) to about 3.00% (w/w), from about 0.05% (w/w) to about 2.50% (w/w), from about 0.05% (w/w) to about 2.00% (w/w), from about 0.05% (w/w) to about 1.50% (w/w), from about 0.05% (w/w) to about 1.25% (w/w), from about 0.05% (w/w) to about 1.00% (w/w), from about 0.050% (w/w) to about 0.75% (w/w), from about 0.050% (w/w) to about 0.500% (w/w), or from about 0.05% (w/w) to about 0.25% (w/w).
In some embodiments, the bulking agent is present in the lyophilized composition from about 0.25% (w/w) to about 3.00% (w/w), from about 0.25% (w/w) to about 2.50% (w/w), from about 0.25% (w/w) to about 2.00% (w/w), from about 0.25% (w/w) to about 1.50% (w/w), from about 0.25% (w/w) to about 1.25% (w/w), from about 0.25% (w/w) to about 1.00% (w/w), from about 0.25% (w/w) to about 0.75% (w/w), or from about 0.25% (w/w) to about 0.50% (w/w). In some embodiments, the bulking agent is present in the lyophilized composition from about 0.50% (w/w) to about 3.00% (w/w), from about 0.50% (w/w) to about 2.50% (w/w), from about 0.50% (w/w) to about 2.00% (w/w), from about 0.50% (w/w) to about 1.50% (w/w), from about 0.50% (w/w) to about 1.25% (w/w), from about 0.50% (w/w) to about 1.00% (w/w), or from about 0.50% (w/w) to about 0.75% (w/w).
In some embodiments, the bulking agent is present in the lyophilized composition from about 0.75% (w/w) to about 3.00% (w/w), from about 0.75% (w/w) to about 2.50% (w/w), from about 0.75% (w/w) to about 2.00% (w/w), from about 0.75% (w/w) to about 1.50% (w/w), from about 0.75% (w/w) to about 1.25% (w/w), or from about 0.75% (w/w) to about 1.00% (w/w). In some embodiments, the bulking agent is present in the lyophilized composition from about 1.00% (w/w) to about 3.00% (w/w), from about 1.00% (w/w) to about 2.50% (w/w), about 1.00% (w/w) to about 2.00% (w/w), from about 1.00% (w/w) to about 1.50% (w/w), or from about 1.00% (w/w) to about 1.25% (w/w).
In some embodiments, the bulking agent is present in the lyophilized composition from about 0.80% (w/w) to about 1.40% (w/w).
In some embodiments, the tonicity agent is selected from mannitol, D-mannitol, trehalose, αα-trehalose dehydrate, sucrose, dextrose, sodium chloride, and maltose. In some embodiments, the tonicity agent is mannitol. In some embodiments, the tonicity agent is D-mannitol. In some embodiments, the tonicity agent is trehalose. In some embodiments, the tonicity agent is αα-trehalose dihydrate. In another embodiment, the tonicity agent is sucrose. In another embodiment, the tonicity agent is dextrose. In another embodiment, the tonicity agent is sodium chloride. In some embodiments, the tonicity agent is maltose.
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.05% (w/w) to about 2.00% (w/w), from about 0.05% (w/w) to about 1.50% (w/w), from about 0.05% (w/w) to about 1.25% (w/w), from about 0.05% (w/w) to about 1.00% (w/w), from about 0.050% (w/w) to about 0.75% (w/w), from about 0.050% (w/w) to about 0.500% (w/w), or from about 0.05% (w/w) to about 0.25% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.25% (w/w) to about 2.00% (w/w), from about 0.25% (w/w) to about 1.50% (w/w), from about 0.25% (w/w) to about 1.25% (w/w), from about 0.25% (w/w) to about 1.00% (w/w), from about 0.25% (w/w) to about 0.75% (w/w), or from about 0.25% (w/w) to about 0.50% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.50% (w/w) to about 2.00% (w/w), from about 0.50% (w/w) to about 1.50% (w/w), from about 0.50% (w/w) to about 1.25% (w/w), from about 0.50% (w/w) to about 1.00% (w/w), or from about 0.50% (w/w) to about 0.75% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.75% (w/w) to about 2.00% (w/w), from about 0.75% (w/w) to about 1.50% (w/w), from about 0.75% (w/w) to about 1.25% (w/w), or from about 0.75% (w/w) to about 1.00% (w/w). In some embodiments, the tonicity agent is present in the lyophilized composition from about 1.00% (w/w) to about 2.00% (w/w), from about 1.00% (w/w) to about 1.50% (w/w), or from about 1.00% (w/w) to about 1.25% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.05% (w/w) to about 10.00% (w/w), from about 0.05% (w/w) to about 6.00% (w/w), from about 0.05% (w/w) to about 5.50% (w/w), from about 0.05% (w/w) to about 5.00% (w/w), from about 0.05% (w/w) to about 4.50% (w/w), or from about 0.05% (w/w) to about 4.00% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.25% (w/w) to about 10.00% (w/w), from about 0.25% (w/w) to about 6.00% (w/w), from about 0.25% (w/w) to about 5.50% (w/w), from about 0.25% (w/w) to about 5.00% (w/w), from about 0.25% (w/w) to about 4.50% (w/w), or from about 0.25% (w/w) to about 4.00% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.50% (w/w) to about 10.00% (w/w), from about 0.50% (w/w) to about 6.00% (w/w), from about 0.50% (w/w) to about 5.50% (w/w), from about 0.50% (w/w) to about 5.00% (w/w), from about 0.50% (w/w) to about 4.50% (w/w), or from about 0.50% (w/w) to about 4.00% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.75% (w/w) to about 10.00% (w/w), from about 0.75% (w/w) to about 6.00% (w/w), from about 0.75% (w/w) to about 5.50% (w/w), from about 0.75% (w/w) to about 5.00% (w/w), from about 0.75% (w/w) to about 4.50% (w/w), or from about 0.75% (w/w) to about 4.00% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 1.00% (w/w) to about 10.00% (w/w), from about 1.00% (w/w) to about 6.00% (w/w), from about 1.00% (w/w) to about 5.50% (w/w), from about 1.00% (w/w) to about 5.00% (w/w), from about 1.00% (w/w) to about 4.50% (w/w), or from about 1.00% (w/w) to about 4.00% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition from about 0.80% (w/w) to about 1.40% (w/w).
In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce an osmolarity equivalent to between about 0.25% saline (w/w) to about 3% saline (w/w) when the lyophilized composition is reconstituted in water. In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce an osmolarity equivalent to between about 1% saline (w/w) to about 2% saline (w/w) when the lyophilized composition is reconstituted in water. In some embodiments, the tonicity agent is present in the lyophilized composition in an amount calculated to produce a hyperosmotic solution. In some embodiments, the hyperosmotic solution has an osmolarity equivalent to greater than 0.9% saline (w/w).
In some embodiments, the antioxidant is selected from methionine, cysteine, histidine, arginine, lysine, and a combination thereof.
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.
In some embodiments, the antioxidant is present in the lyophilized composition from about 0.10% (w/w) to about 15.00% (w/w), from about 0.10% (w/w) to about 10.00% (w/w), from about 0.10% (w/w) to about 7.50% (w/w), from about 0.10% (w/w) to about 5.00% (w/w), from about 0.10% (w/w) to about 4.50% (w/w), from about 0.10% (w/w) to about 4.00% (w/w), from about 0.10% (w/w) to about 3.50% (w/w), from about 0.10% (w/w) to about 3.00% (w/w), from about 0.10% (w/w) to about 2.50% (w/w), from about 0.10% (w/w) to about 2.00% (w/w), from about 0.10% (w/w) to about 1.50% (w/w), from about 0.10% (w/w) to about 1.00% (w/w), or from about 0.10% (w/w) to about 0.50% (w/w).
In some embodiments, the antioxidant is present in the lyophilized composition from about 2.50% (w/w) to about 15.00% (w/w), from about 2.50% (w/w) to about 10.00% (w/w), 2.50% (w/w) to about 7.50% (w/w), from about 2.50% (w/w) to about 5.00% (w/w), from about 2.50% (w/w) to about 4.50% (w/w), from about 2.50% (w/w) to about 4.00% (w/w), from about 2.50% (w/w) to about 3.50% (w/w), or from about 2.50% (w/w) to about 3.00% (w/w). In some embodiments, the antioxidant is present in the lyophilized composition from about 5.00% (w/w) to about 15.00% (w/w), from about 5.00% (w/w) to about 10.00% (w/w), from about 5.00% (w/w) to about 7.50% (w/w).
In some embodiments, the antioxidant is present in the lyophilized composition from about 7.50% (w/w) to about 15.00% (w/w) or from about 7.50% (w/w) to about 10.00% (w/w).
In some embodiments, the antioxidant is present in the lyophilized composition from about 6.00% (w/w) to about 8.00% (w/w).
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 a combination thereof.
In some embodiments, the antimicrobial agent is selected from Penicillin G Potassium (Benzylpenicillin), Penicillin V potassium (Penicillin VK), and a combination thereof.
In some embodiments, the antimicrobial agent is Penicillin G Potassium (Benzylpenicillin). In some embodiments, the antimicrobial agent is Penicillin V.
In some embodiments, the antimicrobial agent is present in the lyophilized composition from about 1.00% (w/w) to about 85.00% (w/w), from about 1.00% (w/w) to about 75.00% (w/w), from about 1.00% (w/w) to about 65.00% (w/w), from about 1.00% (w/w) to about 55.00% (w/w), from about 1.00% (w/w) to about 45.00% (w/w), from about 1.00% (w/w) to about 35.00% (w/w), from about 1.00% (w/w) to about 25.00% (w/w), from about 1.00% (w/w) to about 15.00% (w/w), or from about 1.00% (w/w) to about 5.00% (w/w).
In some embodiments, the antimicrobial agent is present in the lyophilized composition from about 15.00% (w/w) to about 85.00% (w/w), from about 15.00% (w/w) to about 75.00% (w/w), from about 15.00% (w/w) to about 65.00% (w/w), from about 15.00% (w/w) to about 55.00% (w/w), from about 15.00% (w/w) to about 45.00% (w/w), from about 15.00% (w/w) to about 35.00% (w/w), or from about 15.00% (w/w) to about 25.00% (w/w).
In some embodiments, the antimicrobial agent is present in the lyophilized composition from about 3.00% (w/w) to about 85.00% (w/w), from about 3.00% (w/w) to about 75.00% (w/w), from about 3.00% (w/w) to about 65.00% (w/w), from about 3.00% (w/w) to about 55.00% (w/w), from about 30.00% (w/w) to about 45.00% (w/w), or from about 30.00% (w/w) to about 35.00% (w/w).
In some embodiments, the antimicrobial agent is present in the lyophilized composition from about 55.00% (w/w) to about 85.00% (w/w), from about 55.00% (w/w) to about 75.00% (w/w), or from about 55.00% (w/w) to about 65.00% (w/w).
In some embodiments, the antimicrobial agent is present in the lyophilized composition from about 60.00% (w/w) to about 85.00% (w/w), from about 60.00% (w/w) to about 75.00% (w/w), or from about 6.00% (w/w) to about 65.00% (w/w).
In some embodiments, the Streptococcus pyogenes is treated with benzylpenicillin. In some embodiments, the Streptococcus pyogenes is treated with benzylpenicillin and hydrogen peroxide. In some embodiments, the Streptococcus pyogenes is heated following the benzylpenicillin treatment. For example, the benzylpencillin treated Streptococcus pyogenes may be incubated at 30-38° C. for more than 10 minutes, preferably for 10-45 minutes and then further heated at 38-50° C. for 20-60 minutes. An exemplary process for preparing a composition of the present disclosure comprises starting with a main culture of Streptococcus pyogenes; harvesting the Streptococcus pyogenes cells from the main culture; treating the Streptococcus pyogenes cells with hydrogen peroxide; washing the Streptococcus pyogenes cells; resuspending the Streptococcus pyogenes cells in suspension medium; treating the Streptococcus pyogenes cells with benzylpenicillin; heating the Streptococcus pyogenes cells; preparing final suspension of Streptococcus pyogenes cells; and lyophilizing the Streptococcus pyogenes cells.
In some embodiments, the lyophilized composition comprising non-viable Streptococcus pyogenes comprises: non-viable Streptococcus pyogenese (A Group, Type 3) Su strain; maltose, magnesium sulfate, potassium dihydrogen phosphate, 0.9% sodium chloride, methionine, and benzylpenicillin.
Exemplary quantitative formulae for different proposed dosage strengths of an exemplary composition comprising non-viable Streptococcus pyogenes are presented in Table 13A. These compositions are based on the lyophilized product. Exemplary quantitative formulations of the exemplary composition suspended in 0.9% saline are provided in Table 13B.
In some embodiments, the composition comprising non-viable Streptococcus pyogenes comprises intact Streptococcus pyogenes cells.
In some embodiments, viable Streptococcus pyogenes is determined by testing for bacterial growth. In a preferred embodiment, viable Streptococcus pyogenes is detected using a growth test for hemolytic Streptococcus using blood agar.
In some embodiments, the composition comprises no detectable viable Streptococcus pyogenes upon storage for an extended period (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), under various temperature (e.g., from about −15° C. to about 40° C., from about −5° C. to about 30° C., from about 0° C. to about 20° C., from about 0° C. to about 10° C.) and humidity (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).
In some embodiments, the composition comprises no detectable viable Streptococcus pyogenes upon 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.
As used herein, a composition of the disclosure is considered to have “retained potency,” if, after the period specified, the measured potency (as determined by common methods known in the art) of the composition is within a predetermined range of the potency of a suitable reference standard. In some embodiments, potency is calculated with a cytokine release assay. In some embodiments, the range is between 20 and 180% of the reference standard. In some embodiments, the range is between 40 and 160% of the reference standard. In some embodiments, the range is between 50 and 150% of the reference standard. In some embodiments, the range is between 60 and 140% of the reference standard. In some embodiments, the range is between 70 and 130% of the reference standard. In some embodiments, the range is between 80 and 120% of the reference standard. In a preferred embodiment, the range is between 60 and 140% of the reference standard.
In some embodiments, the composition retains potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the composition as described herein.
In some embodiments, the composition retains potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the composition in conditions of between about 2° C. and about 8° C. as described herein.
In some embodiments, the composition retains potency for more than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 26 months, about 28 months, about 30 months, about 32 months, about 34 months, about 36 months, about 38 months, about 40 months, about 42 months, about 44 months, about 46 months, or about 48 months after storage of the composition in conditions of between about 23° C. and about 27° C. and between about 55% and about 65% relative humidity as described herein.
In some embodiments, the potency of the non-viable cells of Streptococcus pyogenes and/or the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the composition is stable, i.e., does not change or changes by not 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%, 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 as described herein.
In some embodiments, the composition comprising non-viable Streptococcus pyogenes is a lyophilized composition or lyophilized formulation. In some embodiments, the composition comprising non-viable Streptococcus pyogenes is a lyophilized powder.
In some embodiments, the composition comprising non-viable Streptococcus pyogenes is OK-432 (Picibanil™, Chugai Pharmaceutical CO., Ltd. Tokyo, Japan). OK-432 is a freeze-dried biological product that is prepared from the Su strain of Streptococcus pyogenes (group A, Type 3) by treatment with benzylpenicillin and heat. OK-432 is not subjected to further treatment, such as isolation, extraction or purification. Bacterial cells remain intact. However, proliferative capacity is lost and Streptococcal infection does not occur when it is administered to humans.
In some embodiments, the composition comprising non-viable Streptococcus pyogenes is developed from the same master cell bank of genetically distinct group A, Type 3 Streptococcus pyogenes as OK-432.
Methods of preparing compositions comprising non-viable Streptococcus pyogenes have been described, for example, in U.S. Pat. Nos. 3,477,914; 3,632,746; Aoki et al., J. Natl. Cancer Inst. 56:687 (1976); each of which is incorporated by reference in its entirety.
In some embodiments, a lyophilized composition as described herein may further be used to prepare a liquid composition of Streptococcus pyogenes. In some embodiments, the liquid composition is a suspension.
In some embodiments, the liquid composition comprises a mixture of a lyophilized composition as described herein and water. In some embodiments, the liquid composition comprises a mixture of a lyophilized composition as described herein and aqueous NaCl solution. In some embodiments, the concentration of sodium chloride is between about 0.5% and about 1.5%, about 0.6% and about 1.4%, about 0.7% and about 1.3%, about 0.8% and about 1.2%, or about 0.8% and 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 the lyophilized composition is 0.9% sodium chloride solution.
In some embodiments, the lyophilized composition is suspended in liquid (e.g., isotonic sodium chloride solution) to prepare a suspension at a concentration of about 0.005 mg/mL to about 0.01 mg/mL.
In certain embodiments, Klinische Einheit (KE) is used as a unit of measurement for doses of the composition comprising non-viable cells of Streptococcus pyogenes. One KE corresponds to 0.1 mg of freeze-dried Streptococci containing approximately 1×108 cells.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount from about 15 KE to about 200 KE, from about 15 KE to about 150 KE, from about 15 KE to about 100 KE, from about 15 KE to about 90 KE, from about 15 KE to about 80 KE, from about 15 KE to about 70 KE, from about 15 KE to about 60 KE, from about 15 KE to about 50 KE, from about 15 KE to about 40 KE, from about 15 KE to about 30 KE, or from about 15 KE to about 20 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount from about 20 KE to about 200 KE, from about 20 KE to about 150 KE, from about 20 KE to about 100 KE, from about 20 KE to about 90 KE, from about 20 KE to about 80 KE, from about 20 KE to about 70 KE, from about 20 KE to about 60 KE, from about 20 KE to about 50 KE, from about 20 KE to about 40 KE, or from about 20 KE to about 30 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount from about 30 KE to about 200 KE, from about 30 KE to about 150 KE, from about 30 KE to about 100 KE, from about 30 KE to about 90 KE, from about 30 KE to about 80 KE, from about 30 KE to about 70 KE, from about 30 KE to about 60 KE, from about 30 KE to about 50 KE, or from about 30 KE to about 40 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount from about 40 KE to about 200 KE, from about 40 KE to about 150 KE, from about 40 KE to about 100 KE, from about 40 KE to about 90 KE, from about 40 KE to about 80 KE, from about 40 KE to about 70 KE, from about 40 KE to about 60 KE, or from about 40 KE to about 50 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount from about 50 KE to about 200 KE, from about 50 KE to about 150 KE, from about 50 KE to about 100 KE, from about 50 KE to about 90 KE, from about 50 KE to about 80 KE, from about 50 KE to about 70 KE, or from about 50 KE to about 60 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the 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.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the 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 the amount of 15±1, ±2, ±3, or ±4 KE, 20±1, ±2, ±3, or +4 KE, 30±1, ±2, +3, or ±4 KE, 40±1, ±2, +3, or ±4 KE, 50±1, ±2, +3, or ±4 KE, 60±1, ±2, +3, or ±4 KE, 70±1, ±2, +3, or ±4 KE, 80±1, ±2, +3, or ±4 KE, 90±1, ±2, 3, or ±4 KE, 100±1, ±2, +3, or ±4 KE, or 150±1, ±2, +3, or ±4 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the 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 Streptococcus pyogenes are present in the lyophilized composition in the amount of 15±1, ±2, ±3, or ±4 KE, 20±1, ±2, +3, or ±4 KE, 30±1, ±2, +3, or ±4 KE, 40±1, ±2, +3, or ±4 KE, 50±1, ±2, +3, or ±4 KE, or 660 1, ±2, +3, or ±4 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of about 15 KE, about 20 KE, about 30 KE, or about 40 KE. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 15±1, ±2, ±3, or +4 KE, 20±1, ±2, ±3, or +4 KE, 30±1, ±2, 3, or ±4 KE, or 40±1, ±2, 3, or ±4 KE.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of at least 3.5% (w/w), at least 3.6% (w/w), at least 3.7% (w/w), at least 3.8% (w/w), at least 3.9% (w/w), at least 4.0% (w/w), at least 4.1% (w/w), at least 4.2% (w/w), at least 4.3% (w/w), at least 4.4% (w/w), at least 4.5% (w/w), at least 4.6% (w/w), at least 4.7% (w/w), at least 4.8% (w/w), at least 4.9% (w/w), at least 5.0% (w/w), at least 5.5% (w/w), at least 6.0% (w/w), at least 6.5% (w/w), at least 7.0% (w/w), at least 7.5% (w/w), at least 8.0% (w/w), at least 8.5% (w/w), at least 9.0% (w/w), at least 9.5% (w/w), or at least 10.0% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of at least 3.5% (w/w), at least 3.6% (w/w), at least 3.7% (w/w), at least 3.8% (w/w), at least 3.9% (w/w), at least 4.0% (w/w), at least 4.1% (w/w), at least 4.2% (w/w), at least 4.3% (w/w), at least 4.4% (w/w), at least 4.5% (w/w), at least 4.6% (w/w), at least 4.7% (w/w), at least 4.8% (w/w), at least 4.9% (w/w), or at least 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), about 5.0% (w/w), about 5.5% (w/w), about 6.0% (w/w), about 6.5% (w/w), about 7.0% (w/w), about 7.5% (w/w), about 8.0% (w/w), about 8.5% (w/w), about 9.0% (w/w), about 9.5% (w/w), or about 10.0% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 15±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 20±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 20±1, ±2, ±3, or ±4 KE, and of about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), or about 4.1% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40±1, ±2, ±3, or ±4 KE, and of about 3.9% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 30±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40±1, ±2, ±3, or ±4 KE, and of about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), or about 4.1% (w/w) of the total weight of the lyophilized composition. In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40±1, ±2, ±3, or ±4 KE, and of about 3.9% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 50±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 60±1, ±2, ±3, or ±4 KE, and of about 3.5% (w/w), about 3.6% (w/w), about 3.7% (w/w), about 3.8% (w/w), about 3.9% (w/w), about 4.0% (w/w), about 4.1% (w/w), about 4.2% (w/w), about 4.3% (w/w), about 4.4% (w/w), about 4.5% (w/w), about 4.6% (w/w), about 4.7% (w/w), about 4.8% (w/w), about 4.9% (w/w), or about 5.0% (w/w) of the total weight of the lyophilized composition.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes increases when the amount of the non-viable cells of Streptococcus pyogenes increases in the lyophilized composition. For example, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE, 30 KE, 20 KE, 15 KE, or 10 KE.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE, 30 KE, 20 KE, or 15 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±3, ±2, or ±1%, 25±3, ±2, or ±1%, 30±3, ±2, or ±1%, 35±3, ±2, or ±1%, 40±3, ±2, or ±1%, ±3, ±2, or ±1%, or 50±3, ±2, or ±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE, 30 KE, 20 KE, or 15 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE, 30 KE, 20 KE, or 15 KE than 10 KE.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 15 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±3, ±2, or ±1%, 25±3, ±2, or ±1%, 30±3, ±2, or ±1%, 35±3, ±2, or ±1%, 40±3, ±2, or ±1%, 45±3, ±2, or ±1%, or 50±3, ±2, or ±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 15 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 15 KE than 10 KE.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 20 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±3, ±2, or ±1%, 25±3, ±2, or ±1%, 30±3, 2, or ±1%, 35±3, 2, or ±1%, 40±3, ±2, or ±1%, 45±3, 2, or ±1%, or 50±3, ±2, or ±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 20 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 20 KE than 10 KE.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 30 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±3, ±2, or ±1%, 25±3, ±2, or ±1%, 30±3, ±2, or ±1%, 35±3, ±2, or ±1%, 40±3, ±2, or ±1%, 45±3, ±2, or ±1%, or 50±3, ±2, or ±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 30 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 30 KE than 10 KE.
In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±3, ±2, or ±1%, 25±3, ±2, or ±1%, 30±3, ±2, or ±1%, 35±3, ±2, or ±1%, 40±3, ±2, or ±1%, 45±3, ±2, or ±1%, or 50±3, ±2, or ±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE than 10 KE. In some embodiments, the relative percentage amount of the non-viable cells of Streptococcus pyogenes in the lyophilized composition is 20±1%, 25±1%, 30±1%, 35±1%, 40±1%, 45±1%, or 50±1% higher when the non-viable cells of Streptococcus pyogenes are present in the lyophilized composition in the amount of 40 KE than 10 KE.
Immune Checkpoint Inhibitors
The methods of the present disclosure provide for administration of a composition comprising non-viable cells of Streptococcus pyogenes and an immune checkpoint inhibitor. An immune checkpoint inhibitor may In some embodiments, an immune checkpoint inhibitor may target PD1/PD-L1/PD-L2 axis, CD80, CD86, B7-H3, B7 H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, PVRL2, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, PVRIG/CD112R, arginase, indoleamine 2,3 dioxygenase (IDO), IL-10, IL-4, IL-1RA, IL-35, or any combination thereof.
In certain embodiments, an immune checkpoint inhibitor is a small molecule, nucleic acid molecule, peptide, protein, antibody or antigen binding fragment thereof, fusion protein, ribozyme, vaccine, or gene editing system. In some embodiments, a nucleic acid molecule is a gene therapy, vaccine, or an inhibitory nucleic acid. In some embodiments, an inhibitory nucleic acid is an antisense oligonucleotide, siRNA, shRNA, or miRNA. In some embodiments, a gene editing system is a CRISPR system, TALEN system, or ZFN system.
In certain embodiments, an immune checkpoint inhibitor targets the PD1/PD-L1/PD-L2 axis. In certain embodiments, an immune checkpoint inhibitor comprises a PD1 inhibitor and/or a PD-L1 inhibitor.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a PD-1 inhibitor, for example a PD-1-specific antibody or binding fragment thereof, such as lambrolizumab, pidilizumab, nivolumab (Opdivo™, formerly MDX-1106), pembrolizumab (Keytruda™, formerly MK-3475), cetrelimab (formerly JNJ 63723283), cemiplimab (Libtayo™), sintilimab (Tyvyt™), tislelizumab (Baizean™), toripalimab (Tuoyi™), penpulimab (formerly AK105), dostarlimab (Jemperli™), camrelizumab (Airuika™ SHR-1210), prolgolimab (formerly BCD 100), pucotenlimab (HX008), serplulimab (HLX10), cadonilimab (Ketanil™, anti-PD1×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 AMP-514), AMP-224, BMS-936558, IAP-0971, IBI-318 (anti-PD1×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.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (Imfinzi™, MEDI4736), atezolizumab (Tecentriq™ RG7446), avelumab (Bavencio™, MSB0010718C), envafolimab (Enweida™, KN035), sugemalimab (Cejemly™), cosibelimab (CK-301), socazolimab (STI A1014), tagitanlimab (HBM 9167 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-PD1×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 tetraspecific antibody), HB-0036 (anti-PD-L1×anti-TIGIT bispecific antibody), HLX-301 (anti-TIGIT×anti-PD-L1 bispecific), or any combination thereof.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a PD-L1 inhibitor, for example a vaccine, such as IO102/IO103 (IDO peptide+PD-L1 peptide vaccine) or mRNA-4359 (IDO peptide+PD-L1 mRNA vaccine).
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes 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.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of CTLA4. In particular embodiments, a modified immune cell is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, tuvonralimab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), cadonilimab (anti-PD1×anti-CTLA4 bispecific), KN-046 (anti-PD-L1×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.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a B7-H4 specific antibody or binding fragment thereof, such as a scFv or fusion protein thereof, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Pat. No. 9,574,000 and PCT Patent Publication Nos. WO 2016/40724 and WO 2013/025779, each of which is incorporated herein in its entirety.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of CD244.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described in, for example, PCT Publication No. WO 2010/084158, which is incorporated herein in its entirety.
In more embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of TIM3.
In still more embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of Gal9.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of A2aR.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015). In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFβ) or Treg development or activity.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an IDO inhibitor, such as levo-1-methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al; Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr. 6-10, 2013), 1-methyl-tryptophan (1-MT)-tira-pazamine, IO102/IO103 (IDO peptide+PD-L1 peptide vaccine), or any combination thereof.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-1-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, MA).
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with a LAIR1 inhibitor.
In certain embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.
Methods of Treatment
Triple Negative Breast Cancer
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.
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 a human adult, adolescent, child, or infant.
In some embodiments, the triple negative breast cancer may be localized, regional, or metastatic. In some embodiments, the triple negative breast cancer may be newly diagnosed or a recurrent cancer.
Triple negative breast cancer may be divided into six distinct subgroups: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR).
In some embodiments, the triple negative breast cancer exhibits complete or partial resistance to a PD1 inhibitor or PD-L1 inhibitor.
A biological sample may be obtained from a subject for determining the presence and/or level of estrogen receptor, progesterone receptor, and HER2, or triple negative status. A “biological sample” as used herein may be a biopsy specimen, blood sample (from which serum or plasma may be prepared), body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. Biological samples may also be obtained from the subject prior to receiving any composition comprising non-viable cells of Streptococcus pyogenes. 
Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until use. The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., parenteral or intravenous administration or formulation.
The composition comprising non-viable Streptococcuspyogenese may be dosed based on milligrams of dried cell mass or KE. Accordingly, references may be made to either mg or KE. In certain embodiments, a dose of the composition comprising non-viable Streptococcus pyogenese is at about 0.1 KE to about 200 KE, about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1KE, 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, a unit dose comprises of the composition comprising non-viable Streptococcus pyogenese is at 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.
In some embodiments, the composition comprising non-viable Streptococcuspyogenese is administered to the subject once a day, twice a week, once a week, biweekly, or once a month. If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol or polyethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.
In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a subject before and after treatment.
In some aspects, the lyophilized pharmaceutical formulation is reconstituted prior to administration, e.g., to form a liquid formulation of the present disclosure.
In some embodiments, the formulation is administered to the subject using conventional modes of delivery including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intrathecal, intramuscular, subcutaneous, or intratumoral administration.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject prior to the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days before the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject concurrently with the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered on the same day as the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject subsequent to the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days after the immune checkpoint inhibitor.
In other embodiments, a method of this disclosure further comprises administering an additional therapy comprising one or more of: an antibody or antigen binding fragment specific for a cancer antigen expressed by the solid tumor being targeted; a small molecule, a chemotherapeutic agent; surgery; radiation therapy treatment; a cytokine; an RNA interference therapy, or any combination thereof.
Exemplary monoclonal antibodies useful in cancer therapies include, for example, monoclonal antibodies described in Galluzzi et al., Oncotarget 5(24):12472-12508, 2014, which antibodies are incorporated by reference in their entirety.
In certain embodiments, a combination therapy method comprises further administering a radiation treatment or a surgery to a subject. Radiation therapy includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer or non-inflamed solid tumor may be used in a subject in combination with a modified immune cell of this disclosure.
In certain embodiments, a combination therapy method comprises further administering a chemotherapeutic agent to a subject. A chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor. Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer 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 disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.
Cytokines can be used to manipulate host immune response towards anticancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42:539, 2015. Cytokines useful for promoting anticancer or antitumor response 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, singly or in any combination. Another cancer therapy approach involves reducing expression of oncogenes and other genes needed for growth, maintenance, proliferation, and immune evasion by cancer cells. RNA interference, and in particular the use of microRNAs (miRNAs) small inhibitory RNAs (siRNAs) provides an approach for knocking down expression of cancer genes. See, e.g., Larsson et al., Cancer Treat. Rev. 16:128, 2017.
In any of the embodiments disclosed herein, any of the therapeutic agents may be administered once or more than once to the subject over the course of a treatment, and, in combinations, may be administered to the subject in any order (e.g., simultaneously, concurrently, or in any sequence) or any combination. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, spread, growth, and severity of the tumor or cancer; particular form of the active ingredient; and the method of administration.
An effective amount of a therapeutic or pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term “therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
Non-Muscle Invasive Bladder Cancer
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.
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 a human adult, adolescent, child, or infant.
Bladder cancer may be classified according to traditional American Joint Committee on Cancer (AJCC) TNM staging. In the absence of nodal (N stage) or distant metastases (M stage), depth of tumor invasion (T stage) is the most important determination to be made and can be dichotomized based on whether the tumor is invading into or beyond the muscularis propia (muscle-invasive bladder cancer, MIBC) or not (non-muscle-invasive bladder cancer, NMIBC). Table B sets forth the staging of primary tumors (T) in bladder cancer according to the AJCC. Tumors may be further classified according to histological grade (low or high). The World Health Organization (WHO)/International Society of Urological Pathology (ISUP) 2004 classification of Non-muscle Invasive Urothelial Neoplasia is provided in Table C. The World Health Organziation (WHO) 2004 Grading System for Urothelial Carcinoma is provided in Table D.







TABLE B







Staging of primary tumors (T) in bladder cancer










Primary 




Tumor Stage
Description







TX
Primary tumor cannot be assessed



Ta
Noninvasive papillary carcinoma



Tis
Carcinoma in situ (CIS)



T1
Tumor invades lamina propria



T2
Tumor invades muscularis propria



T2a
Tumor invades superficial muscularis 




propria (inner half)



T2b
Tumor invades deep muscularis 




propria (outer half)



T3
Tumor invades perivesical tissue/fat



T3a
Tumor invades perivesical tissue/fat 




microscopically



T3b
Tumor invades perivesical tissue fat 




macroscopically (extravesical mass)



T4
Tumor invades prostate, uterus, vagina, 




pelvic wall, or abdominal wall



T4a
Tumor invades adjacent organs 




(uterus, ovaries, prostate stoma)



T4b
Tumor invades pelvic wall and/




or abdominal wall





















TABLE C








2004 World Health Organization/International





Society of Urologic Pathologists: Classification of





Non-muscle Invasive Urothelial Neoplasia





















Hyperplasia (flat and papillary)





Reactive atypia





Atypia of unknown significance





Urothelial dysplasia





Urothelial CIS





Urothelial papilloma





Papillary urothelial neoplasm of low malignant 





potential (PUNLMP)





Non-muscle invasive low-grade (LG) papillary 





urothelial carcinoma





Non-muscle invasive high-grade (HG) papillary 





urothelial carcinoma

















TABLE D





WHO 2004 Grading for Urothelial Carcinoma
















Urothelial papilloma 
Papillary lesion with no abnormal


(completely benign lesion)
histological features



Classified as benign



Very rare but may occur in 



conjunction with UC



Do not recur once resected


Papillary urothelial neoplasm 
Papillary lesion with no cytologic 


of low malignant 
features of malignancy


potential (PUNLMP)
Negligible risk for progression



May recur


Low grade (LG) papillary 
Moderately differentiated 


urothelial carcinoma
papillary lesions



Cytologic features of malignancy 



are present


High grade (HG) papillary 
Poorly differentiated tumors


urothelial carcinoma
Marked cytologic abnormalities









In certain embodiments, the subject having non-muscle invasive bladder cancer has a Ta stage tumor. In certain embodiments, the subject having non-muscle invasive bladder cancer has a T1 stage tumor. In certain embodiments, the subject having non-muscle invasive bladder cancer has a Tis stage (CIS) tumor. In certain embodiments, the CIS tumor may be with or without Ta and/or T1.
In certain embodiments, the subject has a papillary urothelial neoplasm 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.
Non-muscle invasive bladder cancer may also be divided into three distinct risk categories based upon American Urological Association (AUA) and/or the European Association of Urology (EAU) guidelines. The NMIBC risk stratification groups and criteria are provided in Table E.







TABLE E







Definitions of risk stratification groups non-muscle-invasive bladder cancer


according to the American and European Urologic Associations










American Urological
European Urologic


Risk Group
Association (AUA)
Association (EUA)





Low
Solitary LG Ta tumor, <3 cm
Solitary LG Ta tumor, <3 cm



PUNLMP
PUNLMP


Intermediate
Recurrence within 1 year, LG
All tumors not defined as low



Ta
or high risk



Solitary LG Ta, >3 cm




LG Ta, multifocal




HG Ta, ≤3 cm LG T1



High
Carcinoma in situ
Carcinoma in situ



High-grade Tl tumors
Any high-grade tumor



Recurrent or multifocal or large
Any T1 tumor



(>3 cm) high-grade Ta tumors




Any tumor following BCG
Multiple, recurrent, >3 cm



failure
tumors



Lymphovascular invasion or
Highest risk:



non-urothelial histology




High-grade tumor involving
T1 HG with CIS



prostatic urethra





Multiple, large (>3 cm), or




recurrent T1 high-grade tumors




T1 with CIS in prostatic urethra




Some variant histology or




lymphovascular invasion





LG = low grade;


PUNLMP = papillary urothelial neoplasm of low malignant potential;


cHG = high grade;


CIS = carcinoma in situ;


LVI = lymphovascular invasion






In certain embodiments, the subject has non-muscle invasive bladder cancer that has been identified as low risk according to the AUA and/or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that has been identified as intermediate risk according to the AUA and/or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that has been identified as high risk according to the AUA and/or EUA guidelines. In certain embodiments, the subject has non-muscle invasive bladder cancer that has been identified as highest risk according to the EUA guidelines. In certain embodiments, the subject having non-muscle invasive bladder cancer has lymphovascular invasion.
Intravesical Bacillus Calmette-Guerin (BCG) is standard therapy for treating high risk non-muscle invasive bladder cancer for BCG naïve patients, such as following surgical resection or ablation of the tumor. In certain embodiments, the subject having non-muscle invasive bladder cancer has not received prior BCG therapy (BCG naïve). In certain embodiments, the subject having non-muscle invasive bladder cancer has received adequate BCG treatment. In certain embodiments, the subject having non-muscle invasive bladder cancer is unresponsive to BCG therapy. Definitions for BCG-unresponsive disease and adequate BCG treatment is provided in Table F.







TABLE F







Key definitions of BCG-unresponsive non-muscle invasive bladder cancer and


adequate BCG treatment








BCG-unresponsive non-muscle invasive



bladder cancer
Adequate BCG treatment





Persistent or recurrent CIS alone or with Ta/T1
At least five of six doses of the initial


disease within 12 months of adequate BCG
induction course and at least two of three


therapy
doses of the maintenance treatment


Recurrent high-grade Ta/T1 disease within 6
At least five of six doses of the initial


months of completion of adequate BCG therapy
induction course and at least two of six



doses of the second induction course


T1 high-grade disease on the first evaluation



following an induction BCG course









Other terms for describing clinical scenarios where BCG is unsuccessful in treating high risk non-muscle invasive bladder cancer and is no longer a treatment option include, BCG failure (where muscle invasive bladder cancer is detected), BCG-refractory (detection of high-risk lesions during or after adequate treatment at 3 or 6 months of treatment), BCG-relapsing (detection of tumor following initial response after completion of treatment), and BCG inadequately treated (patient did not receive full BCG dose due to intolerance to or unavailability of BCG drug). In certain embodiments, the subject having non-muscle invasive bladder cancer has BCG failure NMIBC. In certain embodiments, the subject having non-muscle invasive bladder cancer has BCG-refractory NMIBC. In certain embodiments, the subject having non-muscle invasive bladder cancer has BCG-relapsing NMIBC. In certain embodiments, the subject having non-muscle invasive bladder cancer was inadequately treated with BCG.
In certain embodiments, the subject having non-muscle invasive bladder cancer has not had a radical cystectomy. In certain embodiments, the subject having non-muscle invasive bladder cancer is ineligible for radical cystectomy.
In some embodiments, the non-muscle invasive bladder cancer may be newly diagnosed or a recurrent cancer.
In some embodiments, the non-muscle invasive bladder cancer exhibits complete or partial resistance to a PD1 or PD-L1 inhibitor.
A biological sample may be obtained from a subject for determining the presence and/or staging or risk level of non-muscle invasive bladder cancer. A “biological sample” as used herein may be a biopsy specimen, blood sample (from which serum or plasma may be prepared), body fluids (e.g., urine, mucosal washings), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. Biological samples may also be obtained from the subject prior to receiving any composition comprising non-viable cells of Streptococcus pyogenes. 
Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until use. The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., parenteral or intravenous administration or formulation.
The composition comprising non-viable Streptococcuspyogenese may be dosed based on milligrams of dried cell mass or KE. Accordingly, references may be made to either mg or KE. In certain embodiments, a dose of the composition comprising non-viable Streptococcus pyogenese is at about 0.1 KE to about 200 KE, about 1 KE to about 100 KE, about 5 KE to about 50 KE, or about 0.1 KE, 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, a unit dose comprises of the composition comprising non-viable Streptococcus pyogenese is at 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.
In some embodiments, the composition comprising non-viable Streptococcuspyogenese is administered to the subject once a day, twice a week, once a week, biweekly, or once a month.
If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol or polyethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.
In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a subject before and after treatment.
In some aspects, the lyophilized pharmaceutical formulation is reconstituted prior to administration, e.g., to form a liquid formulation of the present disclosure.
In some embodiments, the formulation is administered to the subject using conventional modes of delivery including, but not limited to, intravesical, intravenous, intraperitoneal, intraarterial, intrapleural, intrathecal, intramuscular, subcutaneous, or intratumoral administration.
In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject prior to the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days before the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject concurrently with the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered on the same day as the immune checkpoint inhibitor. In some embodiments, the composition comprising non-viable cells of Streptococcus pyogenes is administered to the subject subsequent to the immune checkpoint inhibitor. For example, the composition comprising non-viable cells of Streptococcus pyogenes may be administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days after the immune checkpoint inhibitor.
In other embodiments, a method of this disclosure further comprises administering an additional therapy comprising one or more of: an antibody or antigen binding fragment specific for a cancer antigen expressed by the solid tumor being targeted; a small molecule, a chemotherapeutic agent; surgery; radiation therapy treatment; a cytokine; an RNA interference therapy; a cancer vaccine, or any combination thereof.
Exemplary monoclonal antibodies useful in cancer therapies include, for example, monoclonal antibodies described in Galluzzi et al., Oncotarget 5(24):12472-12508, 2014, which antibodies are incorporated by reference in their entirety.
In certain embodiments, a combination therapy method comprises further administering a radiation treatment or a surgery to a subject. Radiation therapy includes X-ray therapies, such as gamma-irradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer or non-inflamed solid tumor may be used in a subject in combination with a modified immune cell of this disclosure.
In certain embodiments, a combination therapy method comprises further administering BCG therapy to a subject.
In certain embodiments, a combination therapy method comprises further administering a chemotherapeutic agent to a subject. A chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor. Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer 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 disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.
Cytokines can be used to manipulate host immune response towards anticancer activity. See, e.g., Floros and Tarhini, Semin. Oncol. 42:539, 2015. Cytokines useful for promoting anticancer or antitumor response 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, singly or in any combination. Another cancer therapy approach involves reducing expression of oncogenes and other genes needed for growth, maintenance, proliferation, and immune evasion by cancer cells. RNA interference, and in particular the use of microRNAs (miRNAs) small inhibitory RNAs (siRNAs) provides an approach for knocking down expression of cancer genes. See, e.g., Larsson et al., Cancer Treat. Rev. 16:128, 2017.
In any of the embodiments disclosed herein, any of the therapeutic agents may be administered once or more than once to the subject over the course of a treatment, and, in combinations, may be administered to the subject in any order (e.g., simultaneously, concurrently, or in any sequence) or any combination. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, spread, growth, and severity of the tumor or cancer; particular form of the active ingredient; and the method of administration.
An effective amount of a therapeutic or pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term “therapeutic amount” may be used in reference to treatment, whereas “prophylactically effective amount” may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.
Example 1: In Vivo Efficacy of Non-Viable Cells of Streptococcus pyogenes in Monotherapy and in Combination with Anti-mPD-1 Triple Negative EMT6 Breast Cancer Model
Materials and Methods
Animals. 65 female Balb/c mice of 7 weeks of age were purchased from Jackson Laboratories and housed 5 mice per cage, upon arrival, at the in house animal facility with food and water provided ad libitum. Fifty four mice were enrolled in the study upon tumor randomization, the extra 11 mice (injected with tumor cells) were used for randomization purpose only. After one week acclimation, all mice confirmed to be healthy were weighed on Day 0, before implant of tumor cells, and then twice a week until the end of the study.
Treatment. Several days before the study began, Composition 002 (“Comp. 002” or “002”) was received as lyophilized material in individual vials in a cold box. There were 49 vials containing each 20 KE, equivalent each to 2 mg amount of drug. Composition 002 is a lyophilized biological preparation for administration containing non-viable cells of Streptococcus pyogenes (Group A, type 3) Su strain treated with hydrogen peroxide and benzylpenicillin according to the present disclosure. Composition 002 is manufactured using the same master cell bank as OK-432 (Picibanil®).
All vials were kept in the refrigerator (at 4° C.) at the recommended 2-8° C. temperature. Before administration to mice at each treatment day, each vial was dissolved freshly with 1 ml of 0.9% sterile saline solution. Any remaining small dissolved amount from the dosing vial was discarded each time.
The anti-PD1 antibody (CD279, clone RMP1-14, cat #BP0146) and corresponding IgG control (isotype rat IgG2a,k (clone 2A3, cat #BP0089) were purchased from BioXcell. The shipment was delayed a few days, which led to the administration in mice a few days after the scheduled dose time.
Tumor cells and in vitro culture. EMT6 murine triple negative breast cancer cells were thawed from a frozen vial and placed in culture with sterile DMEM medium supplemented with sterile 10% FBS, incubated at 37° C. in a humidified atmosphere of 5% CO2. The cells were passaged in culture at the constant split ratio ˜1:6, which means the cells were consistently harvested at the same confluence (about 70-80%), at exponential phase growth, at each passage, before being re-plated in new T75 culture flasks.
The conditions for culturing and harvesting the cells were kept standardized to minimize any variability. The cells were passaged two days before the scheduled injection in mice. Each time and on the day of implant in mice, the cells were harvested after a brief treatment (1-2 min) with warm trypsin/EDTA solution followed by addition of sterile 10% serum-containing culture medium and two washes in sterile serum-free culture media.
The cell suspension was kept on ice during the preparation, until and during the time of injection to preserve the cell viability and adhesive properties. After the last centrifugation, the cells were counted and resuspended appropriately to the final concentration of 2.0×106/M1 in sterile serum-free culture media for injection of 2×105 cells/mouse. The calculated percent of cell viability was 99% confirming highly viable cells were implanted in mice.
Sample process for flow cytometry (FACS) analysis of SPLCs and TILs: The spleens were collected from the subset of 3 mice in each group under sterile conditions and placed in sterile cold media. A single cell suspension of splenocytes (SPLCs) was prepared by pressing with the plunger of a 3-ml syringe and passing it through 40 um cell strainers. The SPLCs were then processed and stained for FACS analysis.
The tumors were also collected from the same subset of 3 mice in each group of mice and the tumor infiltrating lymphocytes (TILs) were prepared by mechanical dissociation, followed by 30 min treatment with the collagenase D enzyme at the final concentration of 2.5 mg/ml prepared freshly just before use. The cell suspensions were filtered through 70 um cell strainers, washed in sterile HBSS containing 2% FBS, and stained for FACS analysis.
Staining Antibodies and flow cytometry analysis. The antibody panels for flow cytometry and other associated reagents were purchased from BioLegend, BD Bioscience, and ThermoFisher Scientifics, in advance, before the process and staining of samples for FACS analysis.
The immunophenotyping (i.e. including CD8+ T cells, regulatory CD4+ T cells, NK cells, MDSCs, Tregs, and macrophages) and selected stained panels for FACS analysis are described in the Table below.
All samples were analyzed with the LSRFortessa™ (BD Biosciences) flow cytometer. FACS profiles were further analyzed by FloJo software (treeStar).










Staining Panel (Immunophenotyping)








Immune Cell Population
Markers





T cells
CD45+ CD3+


CD4+ T cells
CD45+ CD3+ CD4+


CD8+ T cells
CD45+ CD3+ CD8+


CD8+ T cells
CD45+ CD3+ CD4− CD8+


Tregs
CD3+ CD4+ Foxp3+ CD25+


Granulocytic MDSC
CD45+ CD3− CD11b+ Ly6G+ Ly6Clow


Monocytic MDSC
CD45+ CD3− CD11b+ Ly6G− Ly6Chigh


NK cells
CD45+ CD3− CD49b+ CD335+


M1 macrophages
CD45+ CD3− F4/80+ CD206−


M2 macrophages
CD45+ CD3− F4/80+ CD206+


PD1
PD1+


PD-L1
PD-L1+









Preparation of single cells for scRNA seq.: At the end of the study, three selected tumors were processed for scRNA sequencing: one tumor from the intravenous arm control (Group 2, mouse #4), the second tumor from Composition 002, 10 mg/Kg of the intravenous arm (Group 3, mouse #6), and the third tumor from Composition 002, 10 mg/Kg of the intratumor arm (Group 4, mouse #6).
The tumors were mechanically and enzymatically dissociated to single cells with collagenase type IV (final concentration 1 mg/ml) and DNAse (final concentration 100 units/ml) for ˜1 hr in an incubator at 370 C and 5% CO2 atmosphere. The samples were then gently washed in HBSS containing 2% FBS by centrifugation at 980 rpm, then treated with ACK on ice for 3 minutes, washed again and processed through the cell death removal kit, followed by resuspension in DMEM containing 20% FBS and 10% DMSO for cryopreservation. There are two vials (1 ml each) for each sample containing 2.5×106 cells (vial #1) and 5.0×106 cells (vial #2). All the samples contained single and highly viable cells (average >98%).
The samples were stored cryopreserved until scRNA seq analysis.
Experimental Design and Results
In vivo implant of tumor cells and treatment of tumor bearing mice. One week after acclimation, all 65 anesthetized mice were implanted in the left 4th mammary fat pad with 2×105 EMT tumor cells in the volume of 100 ul. The number of cells injected in this study was reduced compared to the number of 5×105 used an earlier experiment with the purpose to attenuate the rapid formation and growth of tumors, and prevent the early ulceration. This change was worthwhile because it enabled randomizing the mice when the tumor size was ˜100 mm3 prior to treatment initiation and obtain more accurate and less variable efficacy results.
The mice were carefully monitored twice a week and tumor growth was also evaluated twice a week by caliper measurements of length (L) and width (W). The tumor size was calculated with the formula (L×W2)/2). Almost all tumors became caliperable 6 days after the implant and reached their average size of −100 mm3 in 8 days. The tumor take at this time was 97%. The tumor growth data, prior-to randomization, are expressed as average+SEM and represented graphically in FIG. 1.
Eight days after tumor cell injection, 54 mice with tumors of comparable size were randomized into 9 groups with 6 mice/group to ensure the tumor average and standard deviation were similar between the groups before treatment exposure. The remaining 11 mice with tumors too small (or not caliperable) or too large were not enrolled. After randomization, the mice in Group 1 and Group 2 (Controls) received the vehicle saline by the intratumor and intravenous route, respectively. The mice in Group 6 were dosed intraperitoneally (i.p.) with anti-PD-1 antibody (200 ug/mouse, 100 ul volume) as single agent, and mice in the control Groups 1 and 2 received the same dose and volume of IgG control intraperitoneally. The mice in Groups 4 and 8 were dosed intravenously with Composition 002 at the dose 10 mg/Kg (100 ul volume) alone and in combination with anti-PD-1 antibody, respectively.
The mice in the treatment Groups 3, 5, 7, and 9 were dosed with Composition 002 by the intratumoral (i.t.) route at the dose 10 mg/Kg and 20 mg/Kg, as single agent (Group 3 and Group 5) and in combination with anti-PD-1 antibody (Group 7 and Group 9).
The mice in Groups 5 and 9 were originally planned to be dosed intravenously (i.v.) with Composition 002 at the dose of 20 mg/Kg, however this high dose delivered intravenously was not tolerated causing death of one mouse that showed convulsions quickly after being treated. This dose was then reduced to 15 mg/Kg, which however still induced some adverse reactions: reduced activity, rough hair coat, and body weight loss in some mice that resolved within 2-3 days, and more severe symptoms in a few other mice. In a trial with a few extra mice, it was confirmed that 20 mg/Kg of Composition 002 was tolerated when given intratumorally, it was decided then to deliver this dose only intratumorally. Therefore, the mice in Group 5 and Group 9 received first one initial dose of 15 mg/Kg intravenously then continue to be dosed with 20 mg/Kg by the intratumor route.
The treatment schedule of Composition 002 for all mice was twice a week, i.e. Monday and Thursday for 3 weeks. The schedule for anti-PD-1 dosing was also twice a week, one day after treatment with Composition 002. The treatment groups are described in Table 1 below.







TABLE 1







Structure of the Experimental Groups













EMT6 Mouse





No 
Breast Tumor
Treatment
Sacrifice 


Group
of Mice
Cells (mfp)
mg/Kg
Day





1
6
2 × 105
0 (IT)
30 days


2
6
2 × 105
0 (IV)
30 days


3
6
2 × 105
Composition 002
30 days





10 mg/Kg, IT



4
6
2 × 105
Composition 002
30 days





10 mg/Kg, IV



5
6
2 × 105
Composition 002
30 days





20 mg/Kg, IT



6
6
2 × 105
Anti-PD1 Ab
30 days





200 ug/mouse, IP



7
6
2 × 105
Composition 002
30 days





10 mg/Kg, IT+






anti-PD1



8
6
2 × 105
Composition 002
30 days





10 mg/Kg, IV+






anti-PD1



9
6
2 × 105
Composition 002
30 days





20 mg/Kg, IT+






anti-PD1










The volume of the drug delivered intratumorally was 50 ul on the first week and increased to 100 ul thereafter in concomitance to the increased size of the tumors. The volume of 100 ul was kept constant in all other groups during the entire study. It was previously planned to use a multi-side needle (purchased from Cook Medical) to improve the intratumor delivery by better penetration and uniform diffusion of the drug. However, when the needle was used with saline in a trial with two extra mice, its performance was found to be very poor due to the size and thickness of the needle that made hard its insertion without damaging the skin, even though the dimensions were the smallest available.
After treatment initiation, tumor growth continued to be evaluated twice a week by caliper measurements. The data are expressed as tumor volume average+Standard Deviation (STDV) and Standard Error (SEM) and the growth curves for all groups (Average+SEM) are represented graphically in FIG. 2.
The percent tumor growth inhibition (% TGI) in the treatment groups was calculated and the results are reported in Table 2.







TABLE 2







Measure of percent tumor growth inhibition (% TGI) in Balb/c


mice after 3 week of treatment with Composition 002 delivered


intravenously (IV) and intratumorally (IT) (Day 15 and Day 21)












Composition





Treatment
002 Delivery

% TGI
% TGI


Dose
Route
Group No
Day 15
Day 21





Composition 002 IT
IT
3
45
28


10 mg/Kg (n = 6)






Composition 002 IT

5
50
43


20 mg/Kg (n = 6)






Composition 002 IT

7
24
6


10 mg/Kg + anti-






PD1 (n = 6)






Composition 002 IT
IV
9
64
54


20 mg/Kg + anti-






PD1 (n = 6)






Composition 002 IV

4
36
42


10 mg/Kg (n = 6)






Composition 002 IV

8
79
69


10 mg/Kg + anti-






PD1 (n = 6)






Anti-PD-1 IP 200
IP
6
 0
 0


ug/mouse (n = 6)









The tumor efficacy data obtained from the intravenous and intratumor administration of Composition 002 are also shown in separate graphics representations, as average+SEM, in FIGS. 3A and 3B, respectively.
Similar to the results from our earlier efficacy experiment (unpublished), the data in this study demonstrate that Composition 002 at the dose of 10 mg/Kg inhibited EMT6 tumor growth more effectively when it was delivered intravenously than intratumorally. Also, in combination with anti-PD1 antibody, systemic delivery of 10 mg/Kg Composition 002 demonstrated superior anti-tumor activity than the intratumoral administration of either 10 or 20 mg/Kg. Moreover, the intratumor treatment of Composition 002 did not induce a dose response recapitulating the earlier observations. Due to adverse events described above, the higher dose of 20 mg/Kg could not be evaluated for the intravenous delivery route.
Thirty days post-implant of tumor cells, which was Day 21 post-treatment initiation, the 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+anti-PD-1) showed a tumor that measured 130 mm3 on Day 15 post-treatment initiation and gradually decreased in size and regressed becoming not palpable by the end of the study. The spleen was collected from this mouse and analyzed by FACS.
A total of 3 tumors were harvested and dissociated in single cells for scRNA analysis. Interestingly mouse #2 in Group 8 (Composition 002 intravenously+anti-PD-1) showed regression until becoming not caliperable 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. The lungs were collected from all mice in all groups and also fixed in formalin. The details on days of euthanasia and process of samples collected are summarized in Table 3.







TABLE 3







Study termination - Summary














Euthanasia 

Visible 
Other 


Group 
Mouse 
or Death
Sample
Lung
Organs


No
No
Date
Process
Metastasis
Metastases















Group 1
1
Dec. 2, 2021
Fixed tumor,
—
—


Control IT


liver and lungs





2
Dec. 2, 2021
Fixed tumor,
—
—





liver and lungs





3
Nov. 29, 2021
Fixed tumor,
—
—





liver and lungs





4
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





5
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs




Group 2
1
Dec. 2, 2021
Fixed tumor,
—
—


Control IV


liver and lungs





2
Dec. 2, 2021
Fixed tumor,
—
—




(dead)
liver and lungs





3
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





4
Dec. 3, 2021
Tumor for
—
—





scRNA seq







Fixed lungs





5
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs




Group 3
1
Dec. 2, 2021
Tumor and
—
—


Composition


spleen-FACS




002


Fixed lungs




10 mg/Kg IT
2
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





3
Dec. 2, 2021
Fixed tumor,
—
—





liver and lungs





4
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





5
Nov. 29, 2021
Fixed tumor,
—
—





liver and lungs





6
Dec. 3, 2021
Tumor for
—
—





scRNA seq







Fixed lungs







and liver




Group 4
1
Dec. 3, 2021
Tumor and
—
—


Composition


spleen-FACS




002


Fixed lungs




10 mg/Kg IV
2
Dec. 3, 2021
Fixed tumor,
one lung 
—





liver and lungs
met to







confirm by







histology




3
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





4
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





5
Dec. 2, 2021
Fixed tumor,
—
—





liver and lungs





6
Dec. 3, 2021
Tumor for
—
—





scRNA seq







Fixed lungs







and liver




Group 5
1
Nov. 23, 2021
Fixed tumor,
Lung
—


Composition

(dead)
lungs and liver
metastases



002 IT 
2
Dec. 2, 2021
Tumor and
—
—


20 mg/Kg


spleen-FACS







Fixed lungs





3
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver





4
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver





5
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs




Group 6
1
Dec. 3, 2021
Fixed tumor,
—
—


Anti-PD-1 IP


lungs and liver





2
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





3
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





4
Nov. 30, 2021
Fixed tumor,
—
—





lungs and liver





5
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Nov. 29, 2021
Fixed tumor,
—
—





lungs and liver




Group 7
1
Dec. 2, 2021
Tumor and
—
—


Composition


spleen-FACS




002


Fixed lungs




10 mg/
2
Dec. 2, 2021
Fixed tumor,
—
—


Kg IT +


lungs and liver




anti-PD-1
3
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





4
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver





5
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver





6
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs




Group 8
1
Dec. 3, 2021
Fixed tumor,
—
—


Composition


lungs and liver




002
2
Dec. 3, 2021
Fixed mfp
—
—


10 mg/


tissue, lungs




Kg IV +


and liver




anti-PD1
3
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





4
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





5
Dec. 3, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Dec. 1, 2021
Fixed tumor,
—
—




(dead)
lungs and liver




Group 9
1
Dec. 2, 2021
Fixed tumor,
—
—


Composition


lungs and liver




002
2
Dec. 2, 2021
Tumor and
—
—


20 mg/


spleen-FACS




Kg IT +


Fixed lungs




anti-PD-1
3
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver





4
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





5
Dec. 2, 2021
Tumor and
—
—





spleen-FACS







Fixed lungs





6
Dec. 2, 2021
Fixed tumor,
—
—





lungs and liver









There were no eye-visible metastases on the lungs of the mice from all groups except for one with size 1-2 mm on the lung surface of mouse #2 in Group 4 (Composition 002 10 mg/Kg, IV) that, however, will need to be confirmed by histology.
The fixed samples have been processed for paraffin embedding and blocks from all samples were made by a histopathology lab.
A few mice had tumors that grew larger and presented a dark scab as indicator of beginning ulceration, which led to their euthanasia a few days before the end of the study to be in compliance with the IACUC regulations. As shown by the data and compared with our earlier study (unpublished), this time the implant of a lower number of tumor cells resulted in less aggressive tumor growth and reduced number of large and ulcerated tumors. Both tumors and spleens were also weighed and the weigh data are expressed as mean+SEM are reported graphically in FIGS. 4A-4B (intratumor delivery groups) and FIGS. 5A-5B (intravenous delivery groups). Interestingly, an inverse correlation was found in the size between tumors and spleens: mice that showed better response to Composition 002 had reduced tumor growth and larger spleen. This result was more pronounced and statistically significant in the IV delivery groups that showed the best response to Composition 002 at 10 mg/Kg in combination with anti-PD-1 and the most enlarged spleen indicating a more robust systemic immune response.
Monitoring of body weights and health of mice. During the entire study, the mice were carefully monitored daily for any abnormal signs, including respiratory distress, weakness, lethargy, difficult ambulation, and/or body weight change (e.g. loss), large tumors (i.e. with >2,000 size limit), ulcerated tumors, metastatic burden, and death.
The body weights and health information were recorded and provided in excel files weekly. The body weight data are expressed as average and percent change; the graphic representations of these data for all groups are reported as pre-randomization and post-randomization, in FIGS. 6A and 6B and FIGS. 7A and 7B, respectively. Separate graphic representations of the data for the intratumor and intravenous drug delivery groups are shown in FIGS. 7A and 7B, respectively.
None of the mice showed a significant body weight change, i.e. >15 or >20% weight loss, however the accurate measure could not be done in the mice bearing the largest tumors or enlarged spleens because the increased weight of the tumor or spleen compensated in part for the reduced body weight. There were 2 death events, one in mouse #2 in Group 2 Control and the other in mouse #6 in Group 8 (combination Composition 002 10 mg/Kg IV and anti-PD-1 antibody); while the death of the control mouse was likely due to the large tumor with dark scab/ulceration, the reason of death for the other mouse was perhaps due to either a treatment effect or a strong immune response or a combination of both factors.
The mice with tumors that reached or surpassed the size limit of 2,000 mm3 were euthanized within 24-48 hours to allow the planned harvest and analysis, and to be in compliance with the IACUC protocol requirements.
Flow cytometry analysis of Splenocytes (SPLNs) and Tumor-Infiltrating Lymphocytes (TILs). Following the harvest and process of tumors (for TIL,s) and spleens (for SPLCs), the samples were stained and analyzed by flow cytometry to determine the effect of Composition 002 on the different types of immune cells, according to the immune-phenotyping and selected panels described above in the procedures section. The data as individual duplicates for each sample and average values+standard deviation are reported in Tables 4A-7C. The percentage of T cells subsets in spleens and tumor-derived TIL,s were analyzed with the flow cytometer. The flow cytometry data of the spleen from the mouse #2 in the combination Group 8 (Composition 002 10 mg/Kg intravenously+anti-PD-1) that showed tumor regression were similar to the average values of this group in regard to all the immune phenotypes.







TABLE 4A







FACS analysis data of spleens from the intratumor groups

















CD4+ 
CD8+ 
PD1+ 
M1 Macro-
M2 Macro-



Mouse
T cells
T cells
T cells
T cells
phages
phages


Group No
No
(%)
(%)
(%)
(%)
(%)
(%)

















Group 1
4
21.30
55.20
23.00
15.20
40.40
53.80


Control
4
21.60
52.10
23.10
13.50
40.90
52.80


(Saline IT)
5
17.50
52.30
23.20
12.60
32.00
63.60



5
18.70
50.40
21.10
16.50
27.60
68.20



6
24.20
47.50
22.60
13.10
26.00
71.10



6
24.80
47.10
23.20
13.90
28.00
69.10



AVG
21.35
50.77
22.70
14.13
32.48
63.10



STDV
2.90
3.10
0.81
1.46
6.63
7.99


Group 3
1
25.90
50.00
19.20
18.50
60.60
29.50


Composition
1
29.10
48.50
18.70
18.80
66.40
23.90


002 (10
2
28.70
50.60
20.40
18.40
67.50
24.80


mg/Kg IT)
2
30.20
51.00
20.70
18.70
65.80
22.10



4
27.90
50.20
16.80
19.70
68.00
22.30



4
28.30
48.80
16.80
20.30
71.80
20.40



AVG
28.35
49.85
18.77
19.07
66.68
23.83



STDV
1.44%
1.00%
1.69
0.76
3.65
3.17


Group 5
2
22.90
53.90
19.40
16.00
76.00
16.50


Composition
2
23.70
53.80
19.50
16.60
72.30
16.10


002 (20
5
22.70
50.90
18.70
22.00
77.70
17.30


mg/Kg IT)
5
23.00
53.10
18.40
22.40
74.00
19.80



6
18.90
57.80
21.00
18.60
74.90
17.80



6
19.60
58.50
20.70
18.40
75.20
19.40



AVG
21.80
54.67
19.62
19.00
75.02
17.82



STDV
2.02
2.92
1.05
2.68
1.82%
1.51


Group 7
1
20.40
58.90
22.90
11.70
68.90
24.00


Composition
1
19.80
58.30
23.00
15.30
69.00
25.50


002 0.02 (10
3
22.90
57.30
22.50
11.50
61.50
32.00


mg/Kg IT +
3
23.20
56.90
20.90
20.30
64.10
26.80


anti-PD-1)
6
20.00
61.30
20.80
17.90
77.60
16.30



6
20.80
62.10
19.10
24.10
72.90
17.80



AVG
21.18
59.13
21.53
16.80
69.00
23.73



STDV
1.49
2.13
1.54
4.96
5.82
5.85


Group 9
2
19.80
59.50
17.50
19.00
75.50
15.10


Composition
2
19.30
62.20
16.60
16.60
74.20
15.80


002 0.02 (20
4
23.00
58.10
17.20
21.30
75.50
16.50


mg/Kg IT +
4
23.80
57.10
18.00
21.60
70.20
21.90


anti-PD-1)
5
18.00
59.70
15.20
27.40
73.50
16.20



5
18.10
62.00
14.90
28.70
75.90
17.30



AVG
20.33
59.77
16.57
22.43
74.13
17.13



STDV
2.49
2.04
1.26
4.73
2.13
2.45
















TABLE 4B







FACS analysis data of spleens from the intratumor groups















PD1+








Macro-

Granulocytic
Monocytic
Regulatory




phages
NK cells
MDSCs
MDSCs
T cells


Group No
Mouse No
(%)
(%)
(%)
(%)
(%)
















Group 1
4
5.54
1.60
60.80
19.90
9.01


Control
4
3.67
1.45
63.40
17.30
9.38


(Saline IT)
5
2.73
1.29
59.90
19.20
12.40



5
5.35
1.52
59.70
18.30
14.90



6
5.37
1.19
65.20
17.60
9.34



6
5.16
1.01
64.90
17.50
10.10



AVG
4.64
1.34
62.32
18.30
10.86



STDV
1.16
0.22
2.28
0.96
2.13


Group 3
1
10.60
1.70
76.30
12.50
8.96


Composition
1
8.02
2.20
73.70
13.90
10.10


002 (10
2
8.57
1.22
72.90
13.80
8.15


mg/Kg IT)
2
8.85
1.27
73.60
12.70
8.85



4
8.60
2.40
65.70
17.70
9.85



4
8.16
2.52
64.20
18.00
9.53



AVG
8.80
1.89
71.07
14.77
9.24



STDV
0.93
0.57
4.90
2.46
0.72


Group 5
2
6.25
2.10
64.30
19.50
9.78


Composition
2
6.25
2.29
63.80
20.00
10.00


002 (20
5
9.26
1.24
67.00
17.70
8.47


mg/Kg IT)
5
7.91
1.09
69.00
16.40
8.54



6
4.46
0.78
62.90
20.80
10.30



6
5.43
0.76
65.10
18.20
9.35



AVG
6.59
1.38
65.35
18.77
9.41



STDV
1.73
0.66
2.26
1.63
0.76


Group 7
1
5.08
1.06
58.90
22.90
12.00


Composition
1
5.17
0.97
63.80
18.80
12.50


002 0.02 (10
3
6.92
1.35
59.40
20.40
9.77


mg/Kg IT +
3
7.64
1.62
61.70
18.50
10.10


anti-PD-1)
6
7.34
1.75
51.40
26.20
9.51



6
6.01
1.72
55.80
22.50
12.50



AVG
6.36
1.41
58.50
21.55
11.06



STDV
1.10
0.34
4.40
2.92
1.42


Group 9
2
6.51
0.83
57.90
22.30
9.21


Composition
2
5.84
1.03
57.50
23.20
8.99


002 0.02 (20
4
5.36
1.18
45.20
27.00
10.00


mg/Kg IT +
4
4.90
1.02
46.30
26.40
9.42


anti-PD-1)
5
7.89
0.88
54.10
26.00
8.01



5
6.98
0.84
55.90
24.90
8.15



AVG
6.25
0.96
52.82
24.97
8.96



STDV
1.10
0.14
5.65
1.87
0.76
















TABLE 4C







FACS analysis data of spleens from the intratumor groups

















Granulocytes




PD-L1
PD-L1
M1/
MDSCs/


Group 
Mouse 
T Cells
Macrophages
M2
Monocytes


No
No
(%)
(%)
Ratio
MDSCs Ratio















Group 1
4
10.10
3.14
0.75
3.06


Control
4
7.35
2.10
0.77
3.66


(Saline IT)
5
7.84
2.64
0.50
3.12



5
9.51
2.47
0.40
3.26



6
7.99
1.72
0.37
3.70



6
8.37
2.42
0.41
3.71



AVG
8.53
2.42
0.51
3.41



STDV
1.06
0.48
0.83
2.38


Group 3
1
20.20
26.40
2.05
6.10


Composition
1
21.50
22.00
2.78
5.30


002 (10
2
18.20
17.80
2.72
5.28


mg/Kg IT)
2
18.40
17.90
2.98
5.80



4
23.60
22.30
3.05
3.71



4
25.90
26.90
3.52
3.57



AVG
21.30
22.22
2.80
4.81



STDV
3.02
3.94
1.15
2.00


Group 5
2
14.50
10.40
4.61
3.30


Composition
2
15.10
8.12
4.49
3.19


002 (20
5
15.90
21.10
4.49
3.79


mg/Kg IT)
5
16.20
19.30
3.74
4.21



6
12.40
12.00
4.21
3.02



6
12.50
9.81
3.88
3.58



AVG
14.43
13.46
4.21
3.48



STDV
1.65
5.40
1.21
1.39


Group 7
1
13.00
12.30
2.87
2.57


Composition
1
14.50
9.59
2.71
3.39


002 0.02 (10
3
11.60
9.82
1.92
2.91


mg/Kg IT +
3
17.80
14.90
2.39
3.34


anti-PD-1)
6
18.50
22.00
4.76
1.96



6
20.60
18.60
4.10
2.48



AVG
16.00
14.54
2.91
2.71



STDV
3.50
4.98
0.99
1.51


Group 9
2
16.30
13.60
5.00
2.60


Composition
2
14.80
16.30
4.70
2.48


002 0.02 (20
4
20.10
18.00
4.58
1.67


mg/Kg IT +
4
19.70
16.80
3.21
1.75


anti-PD-1)
5
18.90
13.80
4.54
2.08



5
19.30
16.00
4.39
2.24



AVG
18.18
15.75
4.33
2.12



STDV
2.13
1.73
0.87
3.02
















TABLE 5A







FACS analysis data of TILs from the 


intratumor groups (duplicated measures)

















CD4+ 
CD8+ 
PD1+ 
M1
M2




T 
T
T
T
Macro-
Macro-


Group 
Mouse
cells
cells
cells
cells
phages
phages


No
No
(%)
(%)
(%)
(%)
(%)
(%)

















Group 1
4
23.80
38.50
10.40
7.72
32.40
55.30


Control
4
23.20
33.80
9.13
8.49
29.60
52.90


(Saline 
5
32.20
76.00
10.70
5.58
23.10
57.40


IT)
5
32.70
77.70
10.10
4.81
21.70
60.20



6
14.10
32.60
8.02
12.50
39.40
57.00



6
15.70
36.80
3.59
12.20
41.40
55.20



AVG
23.62
49.23
8.66
8.55
31.27
56.33



STDV
7.87
21.50
2.67
3.24
8.14
2.48


Group 3
1
51.80
75.50
15.50
7.97
56.20
32.90


Com-
1
52.10
76.10
15.90
10.60
58.60
27.60


position
2
16.40
71.40
11.00
13.80
57.90
33.30


002 (10
2
15.20
68.80
13.50
10.50
56.00
25.30


mg/Kg 
4
31.20
40.30
8.99
17.40
51.30
33.30


IT)
4
32.20
40.10
8.74
15.90
55.70
29.90



AVG
33.15
62.03
12.27
12.70
55.95
30.38



STDV
16.21
17.12
3.16
3.61
2.55
3.38


Group 5
2
43.00
74.20
16.80
12.10
50.80
34.90


Com-
2
43.10
74.70
17.70
14.80
59.40
28.00


position
5
41.40
49.20
15.10
13.60
63.20
26.40


002 (20
5
39.60
54.50
11.40
10.20
55.40
32.20


mg/Kg 
6
11.30
32.70
13.50
11.80
54.30
40.00


IT)
6
18.20
31.10
8.11
13.70
44.40
47.20



AVG
32.77
52.73
13.77
12.70
54.58
34.78



STDV
14.18
19.12
3.58
1.65
6.58
7.80


Group 7
1
21.30
66.80
17.60
12.60
53.40
37.70


Com-
1
21.60
66.30
18.60
12.90
51.60
39.40


position
3
20.00
67.70
18.00
12.20
64.40
30.30


002 0.02 
3
26.40
67.80
18.30
13.60
61.60
30.80


(10 mg/ 
6
38.60
79.40
15.10
14.60
43.70
48.50


Kg IT +
6
34.40
79.90
15.30
12.40
40.30
50.30


anti-
AVG
27.05
71.32
17.15
13.05
52.50
39.50


PD-1)
STDV
7.75
6.48
1.55
0.90
9.51
8.50


Group 9
2
25.30
43.40
16.60
14.90
53.80
38.50


Com-
2
22.70
43.80
16.90
16.60
59.60
34.90


position
4
63.20
79.80
18.10
11.20
50.80
44.60


002 0.02 
4
66.20
79.80
17.80
11.00
55.50
41.50


(20 mg/ 
5
46.30
78.90
15.50
6.13
53.90
40.80


Kg IT +
5
46.30
77.50
16.20
7.65
52.50
40.80


anti-
AVG
45.00
67.20
16.85
11.25
54.35
40.18


PD-1)
STDV
18.27
18.30
0.98
4.03
3.01
3.25
















TABLE 5B







FACS analysis data of TILs from the 


intratumor groups (duplicated measures)















PD1+

Granu-
Mono-
Regu-




Macro-
NK 
locytic
cytic
latory


Group 
Mouse 
phages
cells
MDSCs
MDSCs
T cells


No
No
(%)
(%)
(%)
(%)
(%)
















Group 1
4
10.10
3.82
11.10
22.90
5.34


Control
4
10.80
3.84
13.90
22.20
6.24


(Saline IT)
5
8.82
4.09
30.10
11.60
4.32



5
12.00
7.38
28.90
7.15
4.81



6
16.70
7.00
54.70
8.76
6.82



6
13.30
6.06
50.60
6.88
4.78



AVG
11.95
5.37
31.55
13.25
5.39



STDV
2.79
1.65
18.10
7.40
0.96


Group 3
1
7.69
5.84
16.70
12.50
1.49


Composition
1
7.64
5.87
17.90
12.40
1.82


002 (10
2
7.94
6.27
65.30
8.95
7.28


mg/Kg IT)
2
11.10
5.45
64.90
6.90
4.09



4
20.10
12.30
10.70
23.60
10.80



4
16.90
12.10
9.73
23.80
16.20



AVG
11.90
7.97
30.87
14.69
6.95



STDV
5.37
3.29
26.71
7.29
5.74


Group 5
2
8.75
4.29
18.70
8.50
3.75


Composition
2
7.52
4.55
15.80
7.79
2.25


002 (20
5
14.30
4.72
57.70
9.62
5.00


mg/Kg IT)
5
16.70
4.05
69.40
4.96
1.90



6
5.56
3.96
77.80
3.70
11.30



6
20.00
3.48
67.40
8.14
5.00



AVG
12.14
4.18
51.13
7.12
4.87



STDV
5.72
0.45
27.03
2.28
3.42


Group 7
1
15.00
2.90
31.90
11.60
8.96


Composition
1
6.77
3.19
50.50
13.90
3.23


002 0.02 (10
3
4.31
3.16
30.10
12.10
8.84


mg/Kg IT +
3
12.60
3.82
16.40
10.30
4.72


anti-PD-1)
6
8.79
4.78
14.90
9.68
5.15



6
13.10
4.18
16.80
16.40
4.94



AVG
10.10
3.67
26.77
12.33
5.97



STDV
4.14
0.72
13.77
2.48
2.37


Group 9
2
12.10
3.37
17.70
19.90
11.20


Composition
2
20.40
3.73
12.00
11.60
4.48


002 0.02 (20
4
6.67
5.43
10.30
12.00
3.37


mg/Kg IT +
4
5.88
5.54
12.00
12.10
3.19


anti-PD-1)
5
5.24
3.00
11.90
10.90
2.93



5
8.23
3.71
58.70
18.30
2.72



AVG
9.75
4.13
20.43
14.13
4.65



STDV
5.76
1.08
18.92
3.90
4.65
















TABLE 5C







FACS analysis data of TILs from the intratumor groups 


(duplicated measures)

















Granulocytes




PD-L1
PD-L1

MDSCs/


Group 
Mouse
T Cells
Macrophages
M1/M2
Monocytes


No
No
(%)
(%)
Ratio
MDSCs Ratio















Group 1
4
24.10
7.12
0.59
0.48


Control
4
24.40
8.01
0.56
0.63


(Saline IT)
5
30.80
8.82
0.40
2.59



5
28.00
7.75
0.36
4.04



6
44.90
4.17
0.69
6.24



6
43.50
6.67
0.75
7.35



AVG
32.62
7.09
0.56
2.38



STDV
9.32
1.61
3.29
2.45


Group 3
1
24.20
11.10
1.71
1.34


Composition
1
24.90
12.50
2.12
1.44


002 (10
2
48.70
9.52
1.74
7.30


mg/Kg IT)
2
54.20
27.80
2.21
9.41



4
40.30
21.10
1.54
0.45



4
39.50
20.20
1.86
0.41



AVG
38.63
17.04
1.84
2.10



STDV
12.20
7.14
0.75
3.66


Group 5
2
25.40
11.20
1.46
2.20


Composition
2
26.90
7.52
2.12
2.03


002 (20
5
35.50
42.90
2.39
6.00


mg/Kg IT)
5
32.20
16.70
1.72
13.99



6
32.70
17.20
1.36
21.03



6
33.80
20.00
0.94
8.28



AVG
31.08
19.25
1.57
7.18



STDV
4.01
12.43
0.84
11.85


Group 7
1
21.70
11.80
1.42
2.75


Composition
1
20.10
11.30
1.31
3.63


002 0.02 (10
3
22.50
19.80
2.13
2.49


mg/Kg IT +
3
29.70
11.80
2.00
1.59


anti-PD-1)
6
44.00
18.70
0.90
1.54



6
43.00
21.20
0.80
1.02



AVG
30.17
15.77
1.33
2.17



STDV
10.85
4.60
1.12
5.55


Group 9
2
31.60
15.50
1.40
0.89


Composition
2
29.20
18.40
1.71
1.03


002 0.02 (20
4
46.30
22.60
1.14
0.86


mg/Kg IT +
4
46.00
22.10
1.34
0.99


anti-PD-1)
5
18.50
11.40
1.32
1.09



5
19.30
7.36
1.29
3.21



AVG
31.82
16.23
1.35
1.45



STDV
12.26
6.04
0.93
4.85
















TABLE 6A







FACS analysis data of spleens from the intravenous groups

















CD4+ 
CD8+ 
PD1+ 
M1
M2




T 
T
T
T
Macro-
Macro-


Group 
Mouse
cells
cells
cells
cells
phages
phages


No
No
(%)
(%)
(%)
(%)
(%)
(%)

















Group 2
3
24.30
63.80
23.50
7.21
20.10
75.80


Control 
3
25.00
60.10
26.30
9.21
24.00
71.90


IV
5
20.10
62.70
22.60
10.90
16.90
79.50



5
19.30
63.40
25.00
8.78
22.10
73.50



6
20.90
63.50
23.00
9.94
21.10
74.80



6
19.50
59.90
25.40
12.10
24.30
73.10



AVG
21.52
62.23
24.30
9.69
21.42
74.77



STDV
2.50
1.77
1.48
1.71
2.75
2.69


Group 4
1
21.20
66.30
18.40
19.90
51.70
44.10


Com-
1
21.10
64.50
17.40
28.10
66.40
24.60


position
3
20.10
59.90
21.30
21.30
64.70
28.80


002
3
19.50
59.30
23.70
18.40
62.00
29.40


10 mg/
5
20.30
63.90
20.00
21.80
62.00
28.30


Kg IV
5
19.60
63.60
19.90
25.00
62.50
28.40



AVG
20.30
62.92
20.12
22.42
61.55
30.60



STDV
0.72
2.74
2.22
3.55
5.13
6.83%


Group 6
2
18.10
59.50
26.80
12.50
34.60
58.30


Anti-
2
19.80
58.20
27.70
11.30
27.60
68.30


PD-1
3
18.00
58.20
24.30
14.60
23.80
72.10


IP (200
3
17.10
56.00
26.10
15.30
27.60
69.60


ug/
5
22.70
59.70
26.20
12.20
25.50
70.80


mouse)
5
21.40
60.90
27.00
11.10
24.60
71.50



AVG
19.52
58.75
26.35
12.83
27.28
68.43



STDV
2.19
1.69
1.16
1.74
3.91%
5.15%


Group 8
3
17.10
61.40
21.00
19.80
65.80
28.00


Com-
3
15.20
61.00
17.30
23.60
62.60
28.60


position
4
22.20
66.00
17.90
24.70
71.30
19.90


002
4
23.20
65.10
18.00
22.90
67.40
24.10


10 
5
21.80
66.00
17.50
25.60
67.30
23.70


mg/Kg
5
22.90
64.80
17.30
24.40
73.50
17.00


IV + 
AVG
20.40
64.05
18.17
23.50
67.98
23.55


anti-
STDV
3.38
2.26
1.42
2.04
3.90
4.51


PD1
2
24.60
69.90
16.40
21.30
67.10
26.50



2
25.50
68.10
19.10
21.50
72.00
20.80



AVG
25.05
69.00
17.75
21.40
69.55
23.65
















TABLE 6B







FACS analysis data of spleens from the intravenous groups















PD1+

Granu-
Mono-
Regu-




Macro-
NK 
locytic
cytic
latory


Group 
Mouse 
phages
cells
MDSCs
MDSCs
T cells


No
No
(%)
(%)
(%)
(%)
(%)
















Group 2
3
9.32
1.33
53.80
20.30
12.30


Control 
3
11.80
1.31
50.50
24.20
13.30


IV
5
12.00
1.68
44.30
26.10
14.70



5
11.00
1.98
41.50
24.10
17.00



6
12.70
2.24
41.00
26.80
15.70



6
14.30
2.03
44.10
26.30
15.20



AVG
11.85
1.76
45.87
24.63
14.70



STDV
1.67
0.39
5.15
2.40
1.69


Group 4
1
13.50
1.32
54.40
22.80
10.80


Com-
1
17.60
1.24
53.00
24.70
9.78


position
3
10.60
1.14
52.80
24.00
14.40


002
3
9.72
1.09
56.50
22.80
13.50


10 mg/Kg
5
12.70
1.05
61.80
19.90
11.70


IV
5
15.50
1.00
65.50
18.80
11.60



AVG
13.27
1.14
57.33
22.17
11.96



STDV
2.96
0.12
5.20
2.33
1.71


Group 6
2
12.10
1.21
60.70
18.70
11.80


Anti-PD-1
2
8.39
1.39
60.80
19.70
12.20


IP (200
3
10.90
1.36
58.00
21.50
17.10


ug/mouse)
3
11.30
1.47
59.10
21.10
16.10



5
11.10
1.37
50.90
23.90
13.30



5
9.35
1.36
59.70
17.70
13.90



AVG
10.52
1.36
58.20
20.43
14.07



STDV
1.38
0.08
3.73
2.22
2.12


Group 8
3
14.20
1.16
66.20
17.90
11.00


Com-
3
12.70
0.93
61.80
16.90
11.30


position
4
13.50
0.88
61.30
19.30
8.80


002
4
13.70
1.10
59.00
20.10
7.96


10 mg/Kg
5
14.30
0.77
59.70
18.90
7.47


IV + anti-
5
13.30
0.88
66.90
18.20
7.34


PD1
AVG
13.62
0.95
62.48
18.55
8.98



STDV
0.59
0.15
3.32
1.13
1.76



2
14.00
1.03
49.80
18.80
6.01



2
11.60
1.13
64.50
17.90
7.55



AVG
12.80
1.08
57.15
18.35
6.78
















TABLE 6C







FACS analysis data of spleens from the intravenous groups

















Granulocytes





PD-L1

MDSCs/




PD-L1
Macro-
M1/
Monocytes



Mouse
T Cells
phages
M2
MDSCs 


Group No
No
(%)
(%)
Ratio
Ratio















Group 2
3
8.86
2.56
0.27
2.65


Control IV
3
7.89
2.60
0.33
2.09



5
7.48
1.72
0.21
1.70



5
5.74
2.95
0.30
1.72



6
6.58
2.80
0.28
1.53



6
8.42
3.99
0.33
1.68



AVG
7.50
2.77
0.29
1.86



STDV
1.17
0.73
1.02
2.14


Group 4
1
18.70
21.50
1.17
2.39


Composition
1
25.00
33.60
2.70
2.15


002
3
19.30
25.80
2.25
2.20


10 mg/Kg IV
3
18.20
20.70
2.11
2.48



5
17.70
28.40
2.19
3.11



5
18.70
27.60
2.20
3.48



AVG
19.60
26.27
2.01
2.59



STDV
2.70
4.78
0.75
2.24


Group 6
2
11.20
6.06
0.59
3.25


Anti-PD-1 IP
2
5.71
1.19
0.40
3.09


(200
3
10.10
2.21
0.33
2.70


ug/mouse)
3
9.03
1.46
0.40
2.80



5
5.53
1.61
0.36
2.13



5
8.27
2.56
0.34
3.37



AVG
8.31
2.52
0.40
2.85



STDV
2.30
1.81
0.76
1.68


Group 8
3
16.10
10.80
2.35
3.70


Composition
3
18.00
15.90
2.19
3.66


002
4
28.70%
31.60
3.58
3.18


10 mg/Kg 
4
26.60
32.20
2.80
2.94


IV + 
5
27.40
34.80
2.84
3.16


anti-PD1
5
27.40
36.50
4.32
3.68



AVG
24.03
26.97
2.89
3.37



STDV
5.48
10.82
0.86
2.94



2
17.10
36.60
2.53
2.65



2
15.50
33.30
3.46
3.60



AVG
16.30
34.95
2.94
3.11
















TABLE 7A







FACS analysis data of TILs from the 


intravenous groups (duplicate measures)

















CD4+ 
CD8+ 
PD1+ 
M1
M2




T 
T
T
T
Macro-
Macro-


Group 
Mouse
cells
cells
cells
cells
phages
phages


No
No
(%)
(%)
(%)
(%)
(%)
(%)

















Group 2
3
32.70
39.30
10.90
10.30
34.10
62.90


Control 
3
32.60
26.10
9.14
15.50
22.30
69.10


IV
5
34.10
47.70
11.70
19.70
12.50
83.40



5
36.20
45.70
13.70
26.80
11.70
84.70



6
52.20
12.60
2.36
23.60
39.20
51.90



6
48.90
15.30
2.23
20.90
34.30
57.60



AVG
39.45
31.12
8.34
19.47
25.68
68.27



STDV
8.76
15.32
4.91
5.88
11.90
13.49


Group 4
1
59.60
4.32
0.94
34.50
35.80
63.00


Com-
1
48.20
9.34
2.71
31.70
38.30
61.40


position
3
59.10
6.22
2.72
19.80
53.20
43.50


002
3
71.80
1.85
0.92
19.80
56.30
40.10


10 mg/
5
40.10
8.89
5.23
56.40
42.50
50.00


Kg IV
5
42.50
11.10
5.79
60.60
46.50
48.80



AVG
53.55
6.95
3.05
37.13
45.43
51.13



STDV
12.10
3.47
2.07
17.66
8.15
9.31


Group 6
2
32.00
42.10
9.78
18.50
22.00
80.50


Anti-
2
31.60
43.60
10.50
13.60
37.00
61.10


PD-1
3
56.50
80.20
16.30
5.24
31.00
69.00


IP (200
3
53.00
78.80
13.80
10.10
28.90
64.40


ug/
5
32.50
69.80
12.10
7.38
29.10
68.60


mouse)
5
36.00
66.40
13.80
10.70
26.20
71.80



AVG
40.27
63.48
12.71
10.92
29.03
69.23



STDV
11.38
16.82
2.41
4.69
5.00
6.69


Group 8
3
34.10
40.80
14.40
23.00
41.00
54.10


Com-
3
29.50
52.30
17.20
29.10
41.80
57.50


position
4
35.40
27.80
7.55
17.30
36.20
57.80


002
4
41.60
22.10
5.68
8.33
41.10
53.90


10 mg/
5
57.80
4.98
1.36
36.40
49.30
40.80


Kg IV + 
5
42.90
10.90
4.01
44.40
45.40
44.50


anti-
AVG
40.22
26.48
8.37
26.42
42.47
51.43


PD1
STDV
9.94
17.88
6.17
13.06
4.45
7.09
















TABLE 7B







FACS analysis data of TILs from the 


intravenous groups (duplicate measures)















PD1+

Granu-
Mono-
Regu-




Macro-
NK 
locytic
cytic
latory


Group 
Mouse 
phages
cells
MDSCs
MDSCs
T cells


No
No
(%)
(%)
(%)
(%)
(%)
















Group 2
3
19.40
1.03
66.90
4.95
8.09


Control 
3
12.80
1.08
71.40
2.96
5.36


IV
5
19.60
2.15
67.50
4.24
7.41



5
17.10
1.70
71.90
3.06
5.38



6
17.30
0.94
68.00
3.43
3.45



6
14.50
1.08
68.70
3.63
5.86



AVG
16.78
1.33
69.07
3.71
5.93



STDV
2.69
0.49
2.09
0.76
1.75


Group 4
1
26.10
1.26
67.90
4.16
11.14


Com-
1
26.90
1.86
66.50
4.02
18.50


position
3
20.40
0.94
62.30
4.20
7.14


002
3
23.40
1.31
62.80
4.27
9.38


10 mg/
5
18.30
1.51
64.30
8.98
15.50


Kg IV
5
17.10
1.50
64.90
8.88
11.10



AVG
22.03
1.40
64.78
5.75
12.13



STDV
4.08
0.31
2.15
2.46
4.16


Group 6
2
8.54
0.94
65.10
1.64
2.70


Anti-PD-
2
5.56
1.09
64.30
1.93
2.46


1 IP 
3
6.90
1.02
79.40
2.19
2.89


(200 ug/
3
8.89
0.96
76.30
2.74
2.84


mouse)
5
9.30
0.77
79.90
2.27
2.07



5
11.70
0.83
79.20
1.82
2.93



AVG
8.48
0.94
74.03
2.10
2.65



STDV
2.11
0.12
7.34
0.39
0.33


Group 8
3
10.70
3.47
29.90
9.10
6.46


Com-
3
10.30
3.51
30.20
8.35
8.12


position
4
16.10
2.02
52.70
5.00
8.18


002
4
13.70
1.72
51.20
4.85
9.09


10 mg/
5
12.50
3.94
47.20
13.60
16.90


Kg IV + 
5
13.40
3.13
49.80
12.20
15.40


anti-
AVG
12.78
2.97
43.50
8.85
10.69


PD1
STDV
2.14
0.89
10.58
3.60
4.34
















TABLE 7C







FACS analysis data of TILs from the intravenous groups 


(duplicate measures)















PD-L1

Granulocytes




PD-L1
Macro-
M1/
MDSCs/



Mouse
T Cells
phages
M2
Monocytes


Group No
No
(%)
(%)
Ratio
MDSCs Ratio















Group 2
3
14.40
5.36
0.54
13.52


Control IV
3
10.30
4.15
0.32
24.12



5
45.90
4.15
0.15
15.92



5
46.30
3.20
0.14
23.50



6
12.50
5.30
0.76
19.83



6
10.40
4.71
0.60
18.93



AVG
23.30
4.48
0.38
18.61



STDV
17.73
0.82
0.88
2.75


Group 4
1
27.60
6.06
0.57
16.32


Composition
1
24.40
4.48
0.62
16.54


002
3
18.50
8.60
1.22
14.83


10 mg/Kg IV
3
14.30
10.20
1.40
14.71



5
30.80
30.00
0.85
7.16



5
15.20
24.80
0.95
7.31



AVG
21.80
14.02
0.89
11.26



STDV
6.81
10.68
0.88
0.87


Group 6
2
14.80
1.22
0.27
39.70


Anti-PD-1 IP
2
10.60
1.85
0.61
33.32


(200
3
11.00
3.45
0.45
36.26


ug/mouse)
3
6.47
6.67
0.45
27.85



5
9.02
4.65
0.42
35.20



5
15.70
3.88
0.36
43.52



AVG
11.27
3.62
0.42
35.28



STDV
3.48
1.97
0.75
18.77


Group 8
3
39.00
22.40
0.75
3.29


Composition
3
44.20
20.50
0.73
3.62


002
4
40.70
4.59
0.63
10.54


10 mg/Kg 
4
30.60
5.81
0.76
10.56


IV + anti-PD1
5
40.90
23.00
1.21
3.47



5
33.30
21.00
1.02
4.08



AVG
38.12
16.22
0.83
4.92



STDV
5.14
8.59
0.63
2.93









The graphic representations of the immune cells percent data, displayed in scatter plots, are shown in FIGS. 9-19. These results compare the two delivery routes of Composition 002 at the dose of 10 mg/Kg dose. The statistical analysis to determine significant differences in the expression of the selected markers between the groups was performed using the unpaired t-test and one-way ANOVA using GraphPad Prism 9.
The results shown in FIGS. 9A-9B are similar to the data from our earlier study, which demonstrate that in both splenocytes and tumor TILs of mice treated intravenously with Composition 002 there was only a minimal or not a statistically significant change in the percent of CD3+ T cells in all the groups as well as in the tumors of mice treated with the drug intratumorally. However, this time there was a consistent and significant increase of T cells in the spleens of mice that received Composition 002 intratumorally. Interestingly, this increase however returned to the baseline level when Composition 002 was combined with anti-PD-1 antibody. Composition 002 delivered intratumorally at the high dose of 20 mg/Kg had no such effect.
The results shown in FIGS. 10A-10B, which illustrate the effects of Composition 002 on the spleens and tumors, are relatively consistent with our earlier observations: there was effect in the spleen after drug delivery by the either route as single agent or in combination with anti-PD-1, and only a trend of about 40-50% reduction of CD4+ T cells induced in the tumors by intravenous administration of the drug. In this study the data demonstrate less variable, better and statistically significant inhibition of CD4+ T cells in the tumors after intravenous administration of Composition 002, which was sufficient to compensate for the increase caused by anti-PD1 in the combination treatment.
As shown in FIGS. 11A-11B, the percent of CD8+ T cells [with CD45+ CD3+ CD4− CD8+ phenotype] was reduced in the spleens and tumors by Composition 002 administered intravenously with the reduction in the spleen being statistically significant when compared with both controls and anti-PD-1 treatment. In the combination group with Composition 002 delivered intravenously, the increasing effect by anti-PD-1 as single agent on the CD8+ T cells was significantly attenuated by Composition 002 resulting in the lower level of immune cells with this phenotype; this low level was comparable to that measured after Composition 002 treatment as single agent. In the tumors of mice treated with Composition 002 intratumorally, there was no such sharp inhibitory effect by Composition 002 single agent but there was a synergism by combined Composition 002 and anti-PD-1 that was statistically significant in comparison to each single agent and the controls.
When the percent of NK cells (gated on CD45+CD3−CD49b+CD335+) was examined in the splenocytes or TILs within all intravenous groups, no significant changes were observed except for a significant decrease by Composition 002 or anti-PD-1 as single agent in the spleen compared to the controls, which was further decreased in the combination group, as shown in FIGS. 12A-12B. In the tumors there was no difference between Composition 002 given intravenously or PD-1 as single agent and the controls but there was a statistically significant increase of NK cells in the combination group.
There was no difference in the percent of NK cells in the spleen between the groups in the intratumor arm but there was a statistically significant reduction by anti-PD1 in the tumors in both single agent and combination groups.
To evaluate the effect of treatments on the immunosuppressive microenvironment of EMT6 tumors, both the separate percent of Granulocytes MDSCs and Monocytes MDSCs and the ratio Granulocytes MDSCs vs. Monocytes MDSCs were calculated from the analysis of the myeloid-derived suppressor cell subpopulations granulocytes and monocytes and the data are shown in FIGS. 13A-13B and FIGS. 14A-14B, respectively. These results demonstrate that in the tumors this ratio was not affected by Composition 002 and was elevated by anti-PD-1 antibody, which correlated with the lack of efficacy by PD-1 inhibitor; however, in the combination groups the ratio decreased dramatically likely due some indirect effect of Composition 002 delivered either intravenously or intratumorally. On the contrary, these effects were not evident in the spleen, in particular Composition 002, given to mice as single by either of the routes, increased the MDSC/Monocytes ratio but it did not when combined with anti-PD-1.
The data analysis of regulatory T cells (Treg) with the phenotype CD45+ CD3−CD4+ CD25+Foxp3+, graphically represented in FIGS. 15A-15B, demonstrate no significant changes in the spleens by Composition 002 upon either the intravenous or intratumor administration but a statistically significant increase in the tumors after the intravenous delivery, and a decrease by ant-PD-1 treatment. The anti-PD-1-induced decrease was reversed by combination with Composition 002.
Next, the tumor associated macrophages (TAMs), gated on CD45+ CD3− F4/F80+ CD206− (M1) or CD206+(M2), were evaluated in the subsets of 3 tumors from all groups. The data represented graphically in FIGS. 16A-16B, describe similar findings derived from our previous study. There was again a consistently significant increase of M1 and decrease of M2 macrophages caused by treatment with Composition 002, in both tumors and spleens, leading to high M1/M2 ratio across all the Composition 002 treatment groups in both the IV and IT arms. This ratio was not altered by inhibition of PD-1 remaining elevated in the combination groups, which resulted in a more effective antitumor activity.
The FACS analysis data of the immune-checkpoint PD-1 expressed in T cells (gated on CD45+ CD3+) are shown in FIGS. 17A-17B. The expression level of PD-1 in T cells was increased by Composition 002 treatment in both splenocytes and TILs upon either the intravenous or intratumor treatment compared to the controls, however it was superior and statistically significant in the spleens compared to the tumors and more robust in the intravenous delivery group. The percent of PD-1 remained significantly lower in either spleens or tumors of mice treated with anti-PD-1 compared to those given Composition 002 although the reduction induced by anti-PD-1 antibody was only statistically significant in the spleen when compared to controls. In the combination groups the expression of PD-1 remained elevated compared to that measured in the single agent groups likely because of the increasing effect of Composition 002.
As shown in FIGS. 18A-18B, the intravenous but not the intratumor delivery of Composition 002 increased the percent of PD1+ macrophages (gated on CD45*, CD3− F4/80+) to a greater extent in the tumors than in the spleen. While the anti-PD-1 antibody had no effect on the percent of PD1 macrophages in the spleens, it reduced it in the tumors with a statistically significance compared to the control baseline in the intravenous arm. If the effects by Composition 002 and anti-PD-1 antibody are not consistent across the arms it might just be because of some differences in the baseline level of PD1+ macrophages in only 3 selected samples between the two delivery arms.
In the combination group of the intravenous arm, the level of PD1+ macrophages remained low in the tumors and statistically different compared to the value measured upon treatment with Composition 002.
The expression of PD-L1 on T-cells (gated on CD45+ CD3+) in the tumors, shown in FIGS. 19A-19B, is higher in the spleen of mice treated intravenously with Composition 002 compared to controls and is not affected by anti-PD-1 antibody. There is a statistically significant difference between controls and anti-PD-1 treated tumors in the intratumor arm.
In the groups treated with Composition 002 and anti-PD-1 in combination, the percentage of PD-L1+ T cells remained higher because of the increasing effect of Composition 002, similarly to the PD-1 expression results in T cells.
Finally, the data analysis of PD-L1 macrophages reported in FIGS. 20A-20B, demonstrates a consistent increase induced by Composition 002 in the spleens and tumors of both the intravenous and intratumor groups and no change by anti-PD-1 antibody. This increase is more robust and statistically significant in the spleens than in the tumors and is maintained in the combination treatment with Composition 002 and anti-PD-1 antibody.
All the flow cytometry analysis data described above are focused on the comparison of one dose of Composition 002 (10 mg/Kg) administered to mice intravenously or intratumorally as single agent and in combination with anti-PD-1 antibody.
In this study, the higher dose of Composition 002 (20 mg/Kg) was also tested by the intratumor route and the data are reported in FIGS. 21A-21K. Mostly, the higher dose of Composition 002 did not have a greater impact on the immune phenotypes analyzed compared to the lower dose, except for inducing a slight increase (˜1.5 fold) of M1/M2 ratio in the T cells in the spleens, which was also statistically significant. Also, the combination of Composition 002 at the dose of 20 mg/Kg with anti-PD-1 was more effective in increasing the M1 macrophages than the combination of Composition 002 at the dose of 10 mg/Kg and anti-PD-1. Similarly, the high dose Composition 002 combined with anti-PD-1 caused a slightly higher increase of the percent PD-1 in T cells in the spleen (statistically significant vs. Control Group 1) compared to the lower dose combined with anti-PD-1. There was no such effect was seen in the tumors by any of the two doses of Composition 002 delivered intratumorally.
CONCLUSION
The findings from this study have demonstrated a significant inhibition of EMT6 tumor growth by Composition 002 as a single agent, which recapitulates the results from our earlier, unpublished study, and a superior anti-tumor activity by combination of Composition 002 and anti-PD-1 antibody.
In agreement with our earlier observations, the intravenous drug delivery in this study was more efficacious than the intratumor administration. Composition 002 given to mice intratumorally at the doses of 10 and 20 mg/Kg did not cause a dose-response effect. The high dose of 20 mg/Kg could not be tested by the intravenous route because it demonstrated to be unsafe. Therefore, unless the penetration and distribution of the drug through the tumor tissue is improved, it remains unknown whether increasing the exposure of the tumors to a higher drug regimen could result in increased efficacy.
The most significant and consistent changes induced by Composition 002 were on tumor associated macrophages (TAMs), with an increase of M1 and decrease of M2 cell types in concomitance with a higher percent of PD-1+/PD-L1+ T cells and PD1+/PD-L1+ macrophages. The increase of M1/M2 ratio, consistent with the paradigm that M1 represents anti-tumor activity while M2 leads to tumor progression, explains one of the mechanisms of anti-tumor activity. As it is reported in the literature, the EMT6 breast tumor model has a weak response to anti-PD-1 antibody, and in this study, it demonstrated to be completely resistant to this immune checkpoint inhibitor. Interestingly, despite the lack of efficacy by anti-PD-1 as a single agent, the combination of Composition 002 and anti-PD-1 antibody further reduced tumor growth compared to single therapies.
The higher expression of PD-1 on T cells in the treatment groups is probably due to the high immune activity stimulated by Composition 002 and a favorable immune microenvironment. However, the anti-PD-1 treatment of EMT6 tumors is not sufficient to block the immunosuppressive microenvironment; therefore Composition 002 might have the potential to be a promising drug for targeting the MDSCs to overcome the resistance to anti-PD-1.
In addition, targeting PD-L1, in addition to PD-1, in combination with Composition 002 might be an even better therapy for EMT6 and ultimately for the triple negative breast cancer in the clinic.
Example 2: In Vivo Efficacy of Non-Viable Cells of Streptococcus pyogenes in Monotherapy And in Combination with Anti-mPD-1 in the Syngeneic Bladder Cancer Model MBT-2 Implanted Subcutaneously in C3H/HeN Mice
The antitumor efficacy of Composition 002, a lyophilized preparation of penicillin-treated Streptococcus pyogenes (group A, type 3, substrain), was evaluated alone and in combination with an antibody that targets programmed cell death protein 1 (PD-1) in the mouse bladder tumor model MBT-2 implanted subcutaneously (s.c.) in immunocompetent C3H/HeN mice. The efficacy experiment was initiated with eight groups of 15 mice each for treatment with lyophilized Streptococcus pyogenes at 2, 1 and 0.5 mg/kg given s.c. daily, either alone or in combination with 5 mg/kg anti-mPD-1 given intraperitoneally (i.p.) twice a week. One group received 5 mg/kg anti-mPD-1 alone and one group received the vehicle for lyophilized Streptococcus pyogenes as a control for reference. Tumor volumes at the beginning of the experiment were in the range of 50-150 mm3. The endpoint of the experiment was reached when the termination criteria of tumor volume exceeding 1,500 mm3 was reached in the first animals. The experiment was ended in two cohorts, such that Groups 2-7 ended on Day 8 and Groups 1 and 8 on Day 10.
Antitumor efficacy of all groups was assessed using the vehicle control group as a reference. Tumor samples taken at termination were used for the downstream analysis of tumor-infiltrating leukocytes (TIL). Using two predefined marker panels, CD4+ and CD8+ T cells, Tregs, granulocytic MDSC, monocytic MDSC, NK cells, and M1/M2 macrophage populations in the tumors of 10 animals per group were assessed by FC analysis. EDTA plasma samples taken during and at the end of the experiment and tumor samples were analyzed to assess changes in various cytokines using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A. These data have been reported separately.
Composition 002 at 0.5, 1 and 2 mg/kg in monotherapy and anti-mPD-1 at 5 mg/kg in monotherapy did not display antitumor activity against the MBT-2 tumor model in this study. Combination of 0.5, 1 or 2 mg/kg Composition 002 with anti-mPD-1 was also not efficacious against the MBT-2 tumor model in this study and no statistically significant differences in tumor volume were observed between any of the test groups and the vehicle control group (Kruskal-Wallis combined with Dunn's post test).
FC analysis of cells isolated from the MBT-2 tumors at the end timepoint showed that the percentage of CD45+ cells was lower in the tumors of all test groups than in the control group. The intragroup variability of the percentage of CD4+ and CD8+ cells was very high. 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 compared to the control group (Kruskal-Wallis combined with Dunn's post test). The percentage of granulocytic MDSC was significantly lower while the percentage of monocytic MDSC was significantly higher in all test groups except the 2 mg/kg Composition 002 monotherapy and the 0.5 mg/kg Composition 002/anti-mPD-1 groups compared to the control. No significant differences between test and control groups were observed for NK cells. The frequency of M1 macrophages was significantly higher while the frequency of M2 macrophages was significantly lower in all test groups except the 0.5 mg/kg Composition 002/anti-mPD-1 group compared to the control.
Minor group mean body weight loss and survival rates of 87-100% after adjustment for animals that exited for tumor-related reasons were observed in this study indicating a good tolerability of the test articles.
The efficacy experiment was set up with eight groups of 15 mice each for treatment with Composition 002 at three dose levels either alone or in combination with anti-mPD-1 as outlined in Table 8. Tumor volumes at the beginning of the experiment were in the range of 50-150 mm3.







TABLE 8







Deisgn of In Vivo Efficacy Experiment














Total Daily
Schedule






Dose
[Dosing 

No. of


Group ID
Treatment
[mg/kg/day]
days]
Route
Animals





1
Control
10 ml/kg
0-9
s.c.
15



Vehicle






2
Composition
2
0-7
s.c.
15



002






3
Composition
1
0-7
s.c.
15



N 002






4
Composition
0.5
0-7
s.c.
15



N 002






5
Anti-mPD-1
5
0,3,7
i.p.
15


6
Composition
2//5
0-7//0, 
s.c.//i.p.
15



002 // Anti-

3, 7





mPD-1






7
Composition
1//5
0-7//0, 
s.c.//i.p.
15



002 // Anti-

3, 7





mPD-1






8
Composition
0.5//5
0-9//0, 
s.c.//i.p.
15



002 // Anti-

3, 7





mPD-1





Vehicle for Composition 002: 0.9% NaCl; vehicle for anti-mPD-1: PBS






The endpoint of the experiment was reached when the termination criteria of tumor volume exceeding 1,500 mm3 was reached in the first animals. The experiment was ended in two cohorts, such that Groups 2-7 ended on Day 8 and Groups 1 and 8 on Day 10.
Tumor samples taken at termination were used for the downstream analysis of tumor-infiltrating leukocytes (TIL). Using two predefined marker panels, CD3+/CD4+ and CD3+/CD8+ T cells, Tregs, granulocytic MDSC, monocytic MDSC, NIK cells, and M1/M2 macrophage populations in the tumors of 10 animals per group were assessed by FC analysis.
EDTA plasma samples taken three days after the initiation of therapy and at termination as well as tumor samples were analyzed to assess changes in various cytokines using the Procarta 36-Plex Mouse Cytokine & Chemokine Panel 1A.
Table 9 is a summary of samples collected in this study.







TABLE 9







Sample Collection












No. of 
Type of 
Time or Time



Group
Animals to
Sample,
Frame After Last
Sample 


ID
be Sampled
Fixation
Treatment
Amount





All
10
EDTA Plasma
Exp. Day 3 after
150 μl blood




(live bleed)
administration of






therapy



All
10
Tumor for FC
At the end of the
1/3




analysis
treatment period



All
10
Tumors SF

1/3




(cytokine






analysis)




All
10
Tumor FFPE

1/3


All
10
EDTA Plasma

Max amount





Terminal plasma samples were split into a 70-μl aliquot and remainder






Antitumor efficacy of all groups was assessed using the vehicle control group as a reference. Tumor growth inhibition was determined by the comparison of RTVs of the test groups with the vehicle control group and is expressed as minimum TIC value in percent.
An overview of the implantation and randomization data is given in Table 10. Individual dosing schedules, efficacy, body weight and survival data are presented in Table 11 and Table 12. Tumor growth curves and FC data are presented in FIGS. 22-24.







TABLE 10







Overview of Experiment

















Date of
Number
Tumors
Date of
Number of
Group
Group


Tumor

Tumor
(Gender)
Impl.
Random-
Random-
Median
Mean


Designation/
Exp.
Im-
of
per
ization
ized
Tumor
Tumor


Passage
No.
plantation
Animals
Animal
(Day 0)
Animals
Volume1
Volume1





MBT-2
S447
29 Jan. 2021
239/
1
9 Feb. 2021
120
77.6-
87.6





female



87.5
89.6





1Range at randomization [mm3]













TABLE 11







Antitumor Efficacy

















Dose 


Minimum 







Level


T/C
Ef-




Group 

[mg/kg/
Schedule

[%]
ficacy
Td
Tq


ID
Treatment 1
day]
[Day]
Route
(Day)2
Rating
[Days]
[Days]










Tumor Model MBT-2















1
Control Vehicle
10 ml/kg
0-9
s.c.
n/a
n/a
1.5
4.3


2
COMPOSITION 
2
0-7
s.c.
100.0 (0)
−
1.3
2.4



002









3
COMPOSITION 
1
0-7
s.c.
100.0 (0)
−
1.2
2.8



002









4
COMPOSITION 
0.5
0-7
s.c.
100.0 (0)
−
0.9
2.5



002









5
Anti-mPD-1
5
0, 3, 7
i.p.
100.0 (0)
−
1.2
3.0


6
COMPOSITION 
2//5
0-7//
s.c.//
 71.6 (7)
−
1.1
3.5



002//Anti-

0, 3, 7
i.p.







mPD-1









7
COMPOSITION 
1//5
0-7//
s.c.//
 98.2 (7)
−
0.9
3.7



002//Anti-

0, 3, 7
i.p.







mPD-1









8
COMPOSITION 
0.5//5  
0-9//
s.c.//
 94.9 (9)
−
1.4
3.9



002//Anti-

0, 3, 7
i.p.







mPD-1





n/a = not applicable;


n.r. = not reached (i.e. group median RTVs always <200%/400%)


Efficacy rating:


+ + + +: T/C <5%;


+ + +: 5% ≤ T/C < 10%;


+ +: 10% ≤ T/C < 25%;


+: 25% ≤ T/C < 50%;


+/−: 50% ≤ T/C ≤ 65%;


−: T/C >65%


1 Vehicle for COMPOSITION 002: 0.9% NaCl; vehicle for anti-mPD-1: PBS


2Minimum T/C values are calculated from mean RTV values.













TABLE 12







Body Weight Loss and Survival Rates





















Maxi-

Euthanasia 









mum

for






Dose

Last
Mean
Over-
Tumor-

Other




Level

Day
BWL
all
Related
Adjusted
Deaths/


Group

[mg/kg/
Schedule
of
[%]
Survival
Reasons
Survival
Euthanasia


ID
Treatment
day]
[Day]
Group
(Day)1
Rate2
(Day)
Rate3
(Day)










Tumor Model MBT-2
















1
Control
10 ml/kg
0-9
10
0.9 (6)
11/
1 × ATV
15/15
—



Vehicle




15
>1500
(100%)









(73%)
mm3











(10) 3 ×











ulcerating 











tumor











(6, 7, 9)




2
COM-
2
0-7
8
2.0 (3)
11/
2 × ATV
15/15
—



POSITION




15
>1500
(100%)




002




(73%)
mm3











(8, 8) 2 ×











ulcerating 











tumor











(6, 7)




3
COM-
1
0-7
8
2.5 (3)
13/
1 × ATV
15/15
—



POSITION 




15
>1500
(100%)




002




(87%)
mm3 (8)











1 ×











ulcerating 











tumor











(6)




4
COM-
0.5
0-7
8
3.9 (3)
11/
1 × ATV
13/15
2 ×



POSITION 




15
>1500
 (87%)
found



002




(73%)
mm3 (8)

dead









1 ×

(8, 8)









ulcerating 











tumor











(5)




5
Anti-mPD-1
5
0, 3, 7
8
1.6 (3)
11/
1 × ATV
15/15
—








15
>1500
(100%)









(73%)
mm3 (8)











3 ×











ulcerating 











tumor











(6, 6, 7)




6
COM-
2//5
0-7//
8
1.7 (3)
9/15
1 × ATV
14/15
1 ×



POSITION 

0, 3, 7


(60%)
>1500
 (93%)
found



002//





mm3 (8)

dead (6)



Anti-mPD-1





4 ×











ulcerating 











tumor











(6, 6, 7, 7)




7
COM-
1//5
0-7//
8
2.6 (3)
12/
1 × ATV
14/15
1 ×



POSITION 

0, 3, 7


15
>1500
 (93%)
found



002//




(80%)
mm3 (8)

dead (5)



Anti-mPD-1





1 ×











ulcerating 











tumor











(3)




8
COM-
0.5//5  
0-9//
10
3.0 (3)
8/15
3 × ATV
13/15
2 ×



POSITION 

0, 3, 7


(53%)
>1500
 (87%)
found



002//





mm3

dead



Anti-mPD-1





(10, 10,

(7, 10)









10) 2 ×











ulcerating 











tumor











(9, 10)





Vehicle for COMPOSITION 002: 0.9% NaCl; vehicle for anti-mPD-1: PBS


1Day on which the minimum mean body weight was recorded when at least 50% remained in the group;


n.r.: not relevant, no body weight loss recorded (i.e. group mean RBWs always >100%).


2Number of animals that would have survived beyond the last experimental day over total number of animals in the group.


3Survival rate adjusted for (i.e. including) all animals that were euthanized for tumor-related reasons and for sample collection.






Materials and Methods










Test Reagents










Total Amount
Concentration


Test Reagent
[mg]1
[mg/ml]





Anti-mPD-1
81.66
8.78


COMPOSITION
84
—


002





1Amount active pharmaceutical ingredient delivered unless stated otherwise
















Handling














Storage



Test Article
Delivery Form
Shipping
Temperature
Comments





Anti-mPD-1
Solution
4° C.
4° C.
Protected 






from






light-


COMPOSITION
Powder
4° C.
4° C.
—


002









Formulation

Vehicle for anti-mPD-1: PBS
Vehicle for COMPOSITION 002: 0.900 NaCl
Test reagents were dissolved in or diluted with the appropriate vehicle on dosing days as indicated in the Table below.

















Con-


Con-



centration
Amount 

centration



Stock 
Stock
Amount 
Dosing



Solution
Solution
Vehicle
Solution


Test Article
[mg/mg]
[μl]
[μl]
[mg/ml]





Anti-mPD-1
8.78
854
14 146
0.5








Concentration





Amount
Dosing 




Amount Test
Vehicle
Solution



Test Article
Article [mg]
[μl]
[mg/ml]
Test Article





COMPOSITION
—
1
 5 000
0.2


002
—
1
10 000
0.1



—
1
20 000
0.05









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

Animals

The animals (female C3H/HeNCrl mice) were shipped from Charles River at the standard age of four to six weeks and an acclimatization period of a minimum of one week was applied on arrival prior to use. Animals were arbitrarily numbered using radio frequency identification transponders (Planet TD) during tumor implantation. Each cage was labeled with a record card indicating all relevant experimental details.
Animals were housed in individually ventilated cages (TECNIPLAST Sealsafe-IVC-System, TECNIPLAST, Hohenpeissenberg, Germany), depending on group size, either in type III or type II long cages. They were kept under a 14 L:10 D artificial light cycle. The temperature inside the cages was maintained at 22±1° C. with a relative humidity of 40-70% and 60-65 air changes/hour in the cage. Dust-free bedding consisting of aspen wood chips with approximate dimensions of 5 mm×5 mm×1 mm (ABEDD, LAB & VET Service GmbH, Vienna, Austria, product code: LTE E-001) and additional nesting material were used. The cages including the bedding and the nesting material were changed weekly. The animals were fed autoclaved Teklad Global Extruded 19% Protein Rodent Diet from Envigo RMS SARL and had access to sterile filtered and acidified (pH 2.5) tap water that was changed twice weekly. Feed and water were provided ad libitum. All materials were autoclaved prior to use.
Where necessary, a nutrient fortified water gel (DietGel Recovery from ClearH2O, Maine, USA) was provided to animal cages and changed every other day.
Tumor Cell Cultivation and Implantation
The bladder tumor xenograft used in this study was derived from a commercially available cell line MBT-2.
Cells were grown at 37° C. in a humidified atmosphere with 5% CO2 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) and passaged at 40-60% using TrypLE Express (Thermo Fisher, #12605-010). Recipient animals were anesthetized by inhalation of isoflurane and received 1×106 tumor cells (100 μl of a suspension in PBS) by subcutaneous (s.c.) injection into the right flank. Cell viability in the cell suspension was determined before and after tumor implantation using the CASY TT Cell Counter (OLS OMNI Life Science GmbH & Co. KG, Bremen, Germany).
Enrollment/Initiation of Experiments
Animals were monitored until the tumor implants reached the study volume criteria of 50-150 mm3 in a sufficient number of animals. Mice were assigned to groups aiming at comparable group median and mean tumor volumes. The process of the assignment to groups (enrollment, stratified randomization) is referred to as randomization in this report. The day of randomization was designated as Day 0 of the experiment.
The time from implantation to randomization at the required tumor volume is expressed in days as Induction time (IT).
Animal Monitoring
Animals were routinely monitored at least twice daily on working days and at least once daily on weekends and public holidays. Routine monitoring included inspections for dead animals, assessment of animal welfare and tumor growth by observation, control of feed and water supply and of technical housing conditions. Any observed or suspected impairment of animal welfare was documented. Observations and possible consequences, e.g. application of euthanasia criteria or measures of veterinary care, are reported with the experimental data in Table 12. Where deemed necessary, a post mortem examination of animals was performed.
Body Weights
Animals were weighed daily for the first week, then three times a week, or daily if body weight loss in excess of 10% was recorded. Relative body weights of individual animals were calculated by dividing the individual body weight on Day x (BWx) by the individual body weight on the day of randomization (BW0) multiplied by 100:



 
  
   
    RBW
    x
   
   [
   %
   ]
  
  =
  
   
    
     
      BW
      x
     
     ⁢
     
      
       ❘
       "\[LeftBracketingBar]"
      
      
       [
       g
       ]
      
     
    
    
     
      BW
      o
     
     [
     g
     ]
    
   
   ×
   100
  
 



Group mean relative body weights (RBW) were calculated for evaluation purposes. Group mean RBW values were used to populate graphs for as long as at least 50% of the animals in a group remained alive.
Individual body weight changes in % were calculated by dividing the body weight change from the day of randomization to Day, (BWx−BW0) by the body weight on the day of randomization (BW0) multiplied by 100.



 
  
   Body
   ⁢
      
   weight
   ⁢
      
   
    
     change
     ⁢
     
       
        
     
     (
     
      Day
      x
     
     )
    
    [
    %
    ]
   
  
  =
  
   
    
     
      BW
      x
     
     -
     
      BW
      o
     
    
    
     BW
     o
    
   
   ×
   100
  
 



Tumor Volumes

The absolute tumor volumes (ATVs) were determined by two-dimensional measurement with a digital caliper (S_Cal EVO Bluetooth, Switzerland) on the day of randomization and then three times weekly. Tumor volumes were calculated according to the formula



 
  
   Tumor
   ⁢
      
   volume
  
  =
  
   
    (
    
     1
     ×
     
      w
      2
     
    
    )
   
   ×
   0.5
  
 



where 1=largest diameter and w=width (perpendicular diameter) of the tumor (in mm).

Relative volumes of individual tumors (individual RTVs) for Day x were calculated by dividing the absolute individual tumor volume on Day x (Tx) by the absolute individual tumor volume of the same tumor on the day of randomization (T0) multiplied by 100:



 
  
   
    RTV
    x
   
   [
   %
   ]
  
  =
  
   
    
     T
     x
    
    
     T
     o
    
   
   ×
   100
  
 



Group mean RTV values were used for drawing growth curves and for treatment evaluation for as long as at least 50% of the animals in a group remained alive.
For calculation of the group mean tumor volumes, the values from all animals that were alive on the day in question were included.
Administration of Therapy
Dosing was performed as described in Table 11. The s.c. therapy was administered into a fold of loose skin over the flank instead of the usual loose skin over the neck to facilitate blood collection after therapy.
The first day of dosing was the day of randomization (Day 0).
Dosing Adjustments
When considerable body weight loss is recorded in efficacy studies the following measures are taken:

    
    
        Daily body weight measurements of individual animals with body weight loss >10%
        No therapy for individual animals with body weight loss >15%
        Facilitated access to feed and water for animals with body weight loss >10%
        Resumption of dosing when individual animals have regained a RBW of ≥90%
    
    


Note, DietGel is supplied to all animals in the group/cage if one of them requires facilitated access to feed and water. Dosing omissions may also be applied under the direction of the responsible veterinarian for any other impairment of animal welfare.
Euthanasia Criteria
According to animal welfare regulations and the relevant SOP of Charles River Discovery Research Services Germany, the following humane endpoints apply to individual animals, irrespective of the experimental status:

    
    
        Tumor volume >1500 mm3 (1200 mm3 before weekends)
        Ulcerating, skin-penetrating tumor
        Dermal necrosis at tumor site >5-8 mm diameter
        Body weight loss >30% on any one measuring day
        Continued body weight loss >20% for more than two days
        Rapid recorded decrease in body weight >20% within two days
        Severe impairment of general condition (apathy, pain, markedly reduced feed and water intake, dyspnea, abnormal habitus or behavior)
    
    


Where individual animals fulfilled euthanasia criteria, sampling was performed ahead of the scheduled time and, if feasible, at the correct time interval after administration of the last applicable dose.
Tumor Samples
Tumors were collected immediately after euthanasia and divided into three parts where possible. The first third was prepared for FC analysis. The second part was snap frozen in liquid nitrogen for cytokine analysis and the third section was transferred to fixative (FFPE samples).
The fixation was performed in 10% neutral buffered formalin for approximately 24 h. The fixative was then replaced by submerging the samples in 70% ethanol for up to seven days. Thereafter, samples were dehydrated by sequentially incubating them in the following solutions: 70% ethanol (two times 0.5 h), 80% ethanol (two times 1 h), 100% ethanol (two times 0.5 h), 100% isopropanol (1.5 h), xylene (two times: 1 h; 1.5 h). Finally, samples were infiltrated by and embedded in paraffin.
Tumor samples were not collected in cases of complete tumor remission or severe ulceration at the tumor site.
For FC analysis, the tumors were cut into 2-4 mm pieces and treated with the Miltenyi mouse tumor dissociation kit following the manufacturer's instructions. Briefly, tumor pieces were incubated with the provided enzyme mix on a gentleMACS Dissociator, the resulting cell suspension was strained though a MACS SmartStrainer (100 μm; Miltenyi, #130-110-917), the strainer was washed, the cells were centrifuged at 300×g for 5 min and the supernatant was discarded.
Cells were resuspended in 1×ACK lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.2-7.4) and incubated for 1-3 min at room temperature. Cells were pelleted by centrifugation at 300×g for 5 min and the supernatant was removed. The cells were washed by resuspension in FC buffer (2% FBS in PBS) and centrifugation at 300×g for 5 min. The supernatant was removed, and the cells were resuspended in FC buffer, counted and processed for FC analysis with 5×105 cells per well.
Blood Samples
Blood was collected by retrobulbar sinus puncture under isoflurane anesthesia.
Plasma was prepared by collecting the blood in standard plasma vials containing EDTA as anticoagulant on ice directly followed by two centrifugations at 2000×g for 5 min at 4° C. Plasma was transferred to a new tube on ice, and samples were stored at −80° C. prior to analysis or shipment.
Flow Cytometry










Antibody Panel A



















Catalogue




Target
Fluorochrome
Channel
Isotype
Clone
No.
Supplier
Target





mCD3e
FITC
BL1
Hamster
145-
553062
BD
mCD3e





IgG1, κ
2C11





mLy-6G
PerCp-Cy5.5
BL3
Rat
1A8
127615
BioLegend
mLy-6G





IgG2a, κ






mCD45
AF700
RL2
Rat
30-F11
103127
BioLegend
mCD45





IgG2b, κ






mCD11b
APC Cy7
RL3
Rat
M1/70
561039
BD
mCD11b





IgG2b, κ






mCD4
eF450
VL1
Rat/
RM4-5
48-0042-
Thermo
mCD4





IgG2a,

82
Fisher






kappa






mLy-6C
BV 605
VL3
Rat IgM,
AL-21
563011
BD
mLy-6C





κ






mCD8
BV650
VL4
Rat
53-6.7
100741
BioLegend
mCD8





IgG2a, κ






mCD25
PE
YL1
Rat
PC61
553866
BD
mCD25





IgG1, λ






mFoxP31
APC
RL1
Rat
FJK-16
17-5773-
eBioscience
mFoxP31





IgG2a,

82







kappa






LD
Zombie Aqua
VL2
—
—
423102
BioLegend
LD





1Intracellular marker
















Antibody Panel B


















Catalogue



Target
Fluorochrome
Channel
Isotype
Clone
No.
Supplier





mCD3e
FITC
BL1
Hamster
145-2C11
553061
BD





IgG1, κ





mCD45
AF700
RL2
Rat IgG2b,
30-F11
103127
BioLegend





κ





mCD11b
APC/Cy7
RL3
Rat (DA)
M1/70
561039
BD





IgG2b, κ





mF4/80
BV421
VL1
Rat IgG2a,
BM8
123137
BioLegend





κ





mCD335
BV605
VL3
Rat IgG2a,
29A1.4
137619
BioLegend





κ





mCD49b
PE
YL1
Rat/IgM,
DX5
12-5971-82
Thermo





kappa


Fisher


mCD2061
APC/Cy7
RL1
Rat/
MR6F3
17-2061-82
Thermo





IgG2b,


Fisher





kappa





LD
Zombie Aqua
VL2
—
—
423102
BioLegend





1Intracellular marker






Flow cytometry (FC) buffer used was 2% FBS in PBS. Fc-block antibody used was CD16/32 purified (2.4G2), 0.5 mg/ml (#553142—BD Biosciences).
Cells were transferred to a 96-well plate (5×105 cells/well). Cells were pelleted by centrifugation of the plates at 400×g for 5 min and the supernatant was removed. Fc-block antibody (10 μl/well of a 1:100 dilution in FC buffer) was added to each well and the plates were incubated for 5 min at room temperature. Then specific antibodies targeting cell surface markers (see Section 5.3.10.1 for antibodies used; antibody panels A and B were stained separately) were added as recommended by the manufacturer in Zombie Aqua Fixable Viability stain (diluted 1:100 in PBS buffer) and the plates were incubated at 4° C. protected from light for 30 min. Cells were washed by the addition of 200 μl FC buffer followed by centrifugation of the plates at 400×g for 5 min, and the supernatant was removed.
For staining of intracellular mouse FoxP3, 200 μl fixation solution (BD Pharmingen #51 9006124) was added to the relevant wells and plates were incubated for 30 min at 4° C. protected from light. Cells were pelleted by centrifugation of the plates at 400×g for 5 min at room temperature and the fixation solution was removed. Cells were washed by resuspension in 200 μl permeabilization solution (BD Pharmingen #51 9006125) prewarmed to 37° C. and centrifugation of the plates at 400×g for 5 min and the permeabilization solution was carefully removed. Fresh permeabilization solution (200 μl) was added to the relevant wells and plates were incubated for 30 min at 37° C. protected from light. Cells were pelleted by centrifugation of the plates at 400×g for 5 min and the permeabilization solution was discarded. Cells were washed once in 200 μl FC buffer and incubated with FoxP3 antibody in 40 μl FC buffer per well. After incubation for 20 min at room temperature in the dark, 200 μl FC buffer was added, the plate was centrifuged at 400×g for 5 min and the FC buffer was removed.
Finally, the cells were resuspended in 200 μl FC buffer, transferred to deep well plates where 200 μl FC buffer was added for analysis with the Attune NXT Acoustic Focusing Cytometer (violet (405 nm)/blue (488 nm)/yellow (561 nm)/red (638 nm) laser configuration).
Data Evaluation
Survival Rates
The survival rate (Table 12) was calculated by counting the number of animals that would have survived beyond the last experimental day of each group and dividing them by the total number of animals in the group. Animals that died or were euthanized on the last day of the group for any other reason than sample collection or termination of the group were not counted as survivors. The adjusted survival rate in Table 2 was calculated by counting all surviving animals including those that were euthanized for tumor-related reasons and dividing them by the total number of animals in the group. The following reasons for euthanasia are classed as tumor-related: 1) tumors fulfilling volume-related euthanasia criteria including accessory tumors and 2) ulcerating tumors. Euthanasia of animals due to symptoms of tumor-induced cachexia is not counted as tumor-related.
Tumor Volume Doubling/Quadrupling Time
Tumor volume doubling and quadrupling time (Td, Tq) for test and control groups is defined as the time interval (in days) required for a group to reach a median RTV of 200% or 400%. Data are presented in Table 11.
Inhibition of Tumor Growth, Test/Control Value in % (Min. T/C Value)
The test versus control value for a particular day (T/Cmean in %) was calculated from the ratio of the mean RTV values of test versus control groups on Day x multiplied by 100.



 
  
   T
   /
   
    
     C
     x
    
    [
    %
    ]
   
  
  =
  
   
    
     mean
     ⁢
        
     
      RTV
      x
     
     ⁢
       
     treated
     ⁢
        
     group
    
    
     mean
     ⁢
     
         
       
     
     ⁢
     
      RTV
      x
     
     ⁢
       
     control
     ⁢
        
     group
    
   
   ×
   100
  
 



The minimum (or optimal) T/Cmean value recorded for a test group during an experiment represents the maximum antitumor efficacy for the respective treatment. Please note that minimum T/Cmean values were calculated including any values generated by using the LOCF methodology.










Group minimum T/Cmean values were used for efficacy 


rating as follows:

















−
Inactive
T/Cmean > 65%


± 
Borderline efficacy
50% ≤ T/Cmean ≤ 65%


+
Moderate efficacy
25% ≤ T/Cmean < 50%


+ +
High efficacy
10% ≤ T/Cmean < 25%


+ + +
Very high efficacy
5% ≤ T/C mean < 10%


+ + + +
Regression
T/C mean < 5%









Flow Cytometry Analysis

Flow cytometry data were analyzed with the FlowJo Data Analysis Software. The software automatically determined the frequency of subpopulations in percent relative to the parent population. FC results are presented as percentage of the corresponding parent population and as total counts for each population. Doublet exclusion was performed according to forward scatter height versus forward scatter area to include only single cells, followed by forward/sideward scatter to determine the leukocyte gate and live/dead discrimination. Fluorescence Minus One (FMO) controls were used to establish correct gating. Further gating was performed as required to assess the populations specified. Information on populations analyzed is illustrated in the tables below.










Panel A T Cells, MDSC










Cell Population
Phenotypic markers







T cells
CD45+CD3+



CD4+ T cells
CD45+CD3+CD4+



CD8+ T cells
CD45+CD3+CD8+



CD8+ T cells
CD45+CD3+CD4−CD8+



Tregs
CD3+CD4+Foxp3+CD25+



Granulocytic MDSC
CD45+CD3−CD11b+Ly6G+Ly6Clow



Monocytic MDSC
CD45+CD3−CD11b+Ly6G−Ly6Chigh




















Panel B NK Cells, Macrophages










Cell Population
Phenotypic markers







NK cells
CD45+CD3−CD49b+CD335+



M1 macrophages
CD45+CD3−F4/80+CD206−



M2 macrophages
CD45+CD3−F4/80+CD206+










Statistical Analysis

For the evaluation of the statistical significance of antitumor efficacy, the non-parametric Kruskal-Wallis test [1] followed by Dunn's method for multiple comparisons [2] was performed.
Individual ATVs of test and control groups were compared on the final day of the study on which all groups were available. Statistical analysis was only carried out if at least 50% of the initially randomized animals still remained in the relevant group. No statistically significant differences in tumor volume between the control group and the test groups were observed.
For the FC data, the Kruskal-Wallis test/Dunn's post test was carried out on the different cell populations comparing the percentages of test groups with the control group. Statistically significant differences between test and control groups are marked in FIGS. 23A-23B.
All p-values <0.05 were considered statistically significant. Statistical calculations were per-formed using R (version 3.1.0; https://www.r-project.org/), where Kruskal-Wallis and Dunn's post-test were implemented according to Hollander and Wolfe [3] or GraphPad Prism bioanalytic software (version 9.0 for Microsoft Windows, GraphPad Software, San Diego, California, USA, https://www.graphpad.com/).
Results and Discussion
Antitumor Efficacy
In this study, the antitumor efficacy of COMPOSITION 002 and anti-mPD-1 was assessed in the syngeneic MBT-2 tumor model implanted in C3H mice. An overview of the experiment is given in Table 10. The efficacy results are summarized in Table 11 and in FIGS. 22A-22B.
COMPOSITION 002 at 0.5, 1 and 2 mg/kg in monotherapy did not display antitumor activity against the MBT-2 tumor model in this study (all min. T/C values=100%).
Anti-mPD-1 at 5 mg/kg in monotherapy did not display antitumor activity against the MBT-2 tumor model in this study (min. T/C value 100%).
Combination of 0.5, 1 or 2 mg/kg COMPOSITION 002 with anti-mPD-1 was also not efficacious against the MBT-2 tumor model in this study (min. T/C values ≥71.6%).
No statistically significant differences in tumor volume were observed between the test groups and the vehicle control group on exp. Day 7, the final day on which all groups were in the study.
Flow Cytometry Analysis
FC analysis of cells isolated from the MBT-2 tumors at the end timepoint on Day 10 (Groups 1 and 8) or Day 8 (Groups 2-7) was carried out. The FC results are presented in FIGS. 23A-23F (percentages) and FIGS. 24A-24F (cell counts). The main observations are listed below.
The percentage of CD45+ cells was lower in the tumors of all test groups than in the control group (FIG. 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 with panel A antibodies. Analysis with panel B antibodies gave similar results but also showed significant differences for the 1 mg/kg and 0.5 mg COMPOSITION 002 monotherapy groups.
The frequency of the CD3+ CD11b− T cell population was significantly elevated in all test groups apart from the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 group compared to the control group. The intragroup variability of the percentage of CD4+ and CD8+ cells was very high and significant differences were only observed for CD4+ cells in the 2 mg/kg COMPOSITION 002 monotherapy group where the frequency was statistically significantly lower compared to the control. 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 (FIG. 23B).
The percentage of granulocytic MDSC was significantly lower in all test groups except the 2 mg/kg COMPOSITION 002 monotherapy and the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 groups while the percentage of monocytic MDSC was significantly higher in all test groups except the 2 mg/kg COMPOSITION 002 monotherapy and the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 groups (FIG. 23C).
No significant differences between test and control groups were observed for NK cells (FIG. 23E). The intragroup variability was higher in the test groups than in the control group.
The frequency of M1 macrophages among F4/80+ cells was significantly higher in all test groups except the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 group while the frequency of M2 macrophages among F4/80+ cells was significantly lower in all test groups except the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 group (FIG. 23F).
Body Weight Change, Survival and Observations
The results are summarized in Table 12 and in FIG. 25.
Minimal body weight loss (BWL) was observed in this study indicating a good tolerability of the test articles. The 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 were found dead in the 0.5 mg/kg COMPOSITION 002 monotherapy group on Day 8 and two in the 0.5 mg/kg COMPOSITION 002/anti-mPD-1 group on Days 7 and 10 resulting in survival rates of 87% after adjustment for animals euthanized for tumor-related reasons. One animal was found dead in the other two combination groups, the 2 mg/kg COMPOSITION 002/anti-mPD-1 and the 1 mg/kg COMPOSITION 002/anti-mPD-1 groups on Days 6 and 5 respectively giving an adjusted survival rate of 93%. Survival was 100% in all remaining groups.
REFERENCES


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


ACRONYMS AND ABBREVIATIONS


    
    
        AT Animal tumor
        ATV Absolute tumor volume
        BW Body weight
        BWL Body weight loss
        FC Flow cytometry
        FFPE Formalin-fixed, paraffin embedded
        i.p. Intraperitoneally
        MDSC Myeloid-derived suppressor cells
        M Mouse/murine/monocytic depending on context
        NK Natural killer cells
        PBS Phosphate-buffered saline
        PD-1 Programmed death 1 receptor
        RBW Relative body weight
        RTV Relative tumor volume
        s.c. Subcutaneously
        SF Snap frozen
        SOP Standard operating procedure
        T/C Test versus control value
        Td Tumor volume doubling time
        TIL Tumor-infiltrating leukocytes
        Tq Tumor volume quadrupling time
    
    


Example 3: Preparation of Lyophilized Composition Comprising Non-Viable Streptococcus pyogenes for Injection
Streptococcus pyogenes (A Group, Type 3, Su strain) are cultured in appropriate culture media. After appropriate cultivation period the bacteria are collected by centrifugation, washed, resuspended, and treated with hydrogen peroxide to kill the bacteria. The killed bacteria are centrifuged, washed, and resuspended in suspension medium, such as Berheimers basal medium (BBM) and filtered. The bacterial suspension is treated with benzylpenicillin and heated at 37° C. for ˜10-45 min and at 45° C. for ˜20-60 min. The final bulk suspension is prepared. Vials are filled with the final bulk suspension and lyophilized. The quantitative formulae for different proposed dosage strengths of an exemplary composition comprising non-viable Streptococcus pyogenes are presented in Table 13A. These compositions are based on the lyophilized product. All vial strengths are filled with the same volume (0.41 mL) of suspension before lyophilization.







TABLE 13A







Quantitative Formula for Different Dosage Strengths 


for Exemplary Composition 002









Content per Vial at Different Dosage Strengths














0.02 mg
0.05 mg
0.1 mg
0.3 mg
0.5 mg
0.7 mg


Composition
(0.2 KE)
(0.5 KE)
(1 KE)
(3 KE)
(5 KE)
(7 KE)
















Non-viable
 0.02 mg
 0.05 mg
 0.1 mg
 0.3 mg
 0.5 mg
 0.7 mg


Streptococcus
 (0.21%)
 (0.49%)
 (0.88%)
 (1.93%)
 (2.54%)
 (2.93%)


pyogenes








(dried cell








mass basis)








Maltose
 9.10 mg
 8.90 mg
 8.58 mg
 7.28 mg
 5.99 mg
 4.69 mg



(94.31%)
(86.65%)
(75.77%)
(46.95%)
(30.39%)
(19.63%)


Magnesium 
 0.02 mg
 0.05 mg
 0.10 mg
 0.29 mg
 0.49 mg
 0.68 mg


sulfate
 (0.20%)
 (0.48%)
 (0.86%)
 (1.89%)
 (2.48%)
 (2.86%)


Potassium 
 0.10 mg
 0.24 mg
 0.49 mg
 1.46 mg
 2.44 mg
 3.42 mg


dihydrogen
 (1.01%)
 (2.38%)
 (4.31%)
 (9.44%)
(12.39%)
(14.30%)


phosphate








Sodium 
0.006 mg
0.015 mg
 0.03 mg
 0.09 mg
 0.15 mg
 0.21 mg


chloride 0.9%
 (0.06%)
 (0.15%)
 (0.26%)
 (0.58%)
 (0.76%)
 (0.88%)


(normal saline)








Methionine
 0.04 mg
 0.10 mg
 0.20 mg
 0.61 mg
 1.02 mg
 1.43 mg



 (0.42%)
 (1.00%)
 (1.81%)
 (3.96%)
 (5.20%)
 (6.00%)


Penicillin G 
 0.36 mg
 0.91 mg
 1.82 mg
 5.47 mg
 9.11 mg
12.75 mg


Potassium
 (3.78%)
 (8.87%)
(16.09%)
(35.24%)
(46.24%)
(53.39%)


(Benzylpenicillin)a





aBased on 1667 penicillin units/mg


KE = klinische einheit, defined as clinical unit, corresponding to 0.1 mg non-viable Streptococcus pyogenes dried cell mass






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







TABLE 13B







Example of Exemplary Composition Excipients After 


Suspension with 0.9% Saline













0.2 mg 





Suspension in 





0.9%



Quantity 
Quantity per
saline at 0.01



per viala
0.2 mg Dose
mg/mL 


Component
(mg)
(mg)
(% w/v)













Non-viable Streptococcus
0.1 
0.2
0.002%


pyogenes (dried cell mass)





Benzyl penicillin potassiumb
1.82 mg 
3.64 mg 
0.036%


(Penicillin G potassium)
(3034
(6068




units)
units)



Methionine
0.20
0.40
0.004%


Maltose
8.58
17.16
 0.17%


Magnesium sulfate
0.10
0.20
0.002%


Potassium dihydrogen
0.49
0.98
 0.01%


phosphatec





Sodium Chloride
0.03
0.06
 0.90%


Water
NA
NA
q.s. to 100%





aBased on a 0.1 mg (1 KE) vial


bFor penicillin, there are 1667 units per mg


cThe IID name is monobasic potassium phosphate


KE = klinische einheit, defined as clinical unit, corresponding to 0.1 mg non-viable Streptococcus pyogenes;


NA = not applicable;


q.s. = quantity sufficient;






For dosing, the lyophilized powder is suspended in an isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005-0.01 mg/mL. The volume of product delivered to a subject at this concentration may vary.
Example 4: T Cell Activation By Composition Comprising Non-Viable Streptococcus Pyogenes 
T-cells were isolated from Peripheral Blood Mononuclear Cell (PBMC-two healthy donors) using RapidSheres magnetic beads. T-cells (500,000) were seeded in 96-well plates. T-cells were treated with 0.2 and 0.8 KE/mL of Composition 002 for 72 hours. T-cells and supernatant were collected for analyses of immune checkpoint biomarkers and cytokines (see table below) by FACS and ELISA, respectively.














Targets
Fluorophore









hCD4
BUV395



hCD8
FITC



hPD-1
PE-Cy7



hCTLA-4
BV421



hTIGIT
BV711



hTIM-3
BV605



hLAG-3
APC



hKi67
efluor506



hFoxP3
PE



Live/Dead dye
efluor780










As shown in FIG. 26, COMPOSITION 002 treatment does not change the number of CD4+ and CD8+ T cells. COMPOSITION 002 induces expression of immune checkpoint molecules CTLA4, PD-1, LAG3, TIM3, and TIGIT in CD4+ T cells (FIG. 27A) and CD8+ T cells (FIG. 27B).
Example 5: In Vivo Efficacy of Non-Viable Cells of Streptococcus pyogenes in Monotherapy And in Combination with Anti-mPD-1 in the Orthotopic Bladder Model (MB49 Bladder Cancer Cells)
The antitumor efficacy of a lyophilized preparation of penicillin-treated Streptococcus pyogenes (group A, type 3, substrain), is evaluated alone and in combination with an antibody that targets programmed cell death protein 1 (PD-1) in the mouse orthotopic bladder tumor model (MB49 bladder cancer cells). The study design for monotherapy is shown in Table 6 below. Animals are randomized into study groups by tumor associated bioluminiescence. The lyophilized preparation of penicillin-treated Streptococcus pyogenes composition is dosed once a week for 4 weeks. The mice are observed post-treatment for 1 week. Mice are examined daily, 5 days a week. Body weight is measured twice a week. Bioluminescent imaging (BLI) is obtained 1-2 times weekly for 4 weeks during in-life phase. Optimal dose of lyophilized preparation of penicillin-treated Streptococcus pyogenes for combination study with anti-PD-1 antibody is selected.







TABLE 14







Study Design for mouse orthotopic bladder tumor model (MB49) treated with


preparation of penicillin-treated Streptococcus pyogenes
















Dosing Schedule







Dose
(post-
Group
Route of



Group
Treatment
(KE/ml)
implantation)
Size
Administration
Endpoints





1
No treatment
NA
NA
10
NA
Body weights


2
Vehicle (saline)
0
Day 7, 14, 21, 28
10
Intrabladder
Survival rate


3
Treatment 
0.8
Day 7, 14, 21, 28
10
Intrabladder
BLI of primary



dose 1




tumor


4
Treatment 
1.6
Day 7, 14, 21, 28
10
Intrabladder
(2x/week; last



dose 2




measurement


5
Treatment 
3.2
Day 7, 14, 21, 28
10
Intrabladder
right before



dose 3




takedown) Bladder








measurements,








digital pictures,








bladder weights








Keep bladder








samples in 10%








formalin at








takedown 4-5








days after last








treatment (Day








29 or 30) for








further








biomarker








analysis









The study design for combination therapy is shown in Table 15 below. Animals are randomized into study groups by tumor associated bioluminiescence. The lyophilized preparation of penicillin-treated Streptococcus pyogenes composition is dosed once a week for 4 weeks. Nice receive anti-PD-1 antibody twice a week. The mice are observed post-treatment for 1 week. Mice are examined daily, 5 days a week. Body weight is measured twice a week. Bioluminescent imaging (BLI) is obtained 1-2 times weekly for 4 weeks during in-life phase.







TABLE 15







Study Design for mouse orthotopic bladder tumor model (MB49) treated with


lyophilized preparation of penicillin-treated Streptococcus pyogenes and anti-PD-1 antibody


















Anti-







S.
Dosing
PD-1
Dosing






pyogenes
Schedule
IgG dose
Schedule





Treat-
Dose
(post
(mg/
(post
Group



Group
ment
(KE/ml)
implantation)
mouse)
implantation)
Size
Endpoints

















1
No
NA
NA
0
Biweekly
10
Body weights



treatment





Survival rate









BLI of









primary tumor


2
IgG
0
Day 7, 14, 
0.2
Biweekly
10
(2x/week; last



control

21, 28



measurement









right before









takedown)


3
S.
Optimal
Day 7, 14, 
0
Biweekly
10
Bladder



pyogenes
dose
21, 28



measurements, 









digital









pictures,


4
Anti-PD-1
NA
Day 7, 14, 
0.2
Biweekly
10
bladder





21, 28



weights









Keep bladder









samples in


5
S.
Optimal
Day 7, 14, 
0.2
Biweekly
10
10% formalin



pyogenes + 
dose
21, 28



at takedown



Anti-





4-5 days after



PD-1





last treatment









(Day 29 or 30)









for further









biomarker









analysis









Example 6: Preparation of Lyophilized Composition Comprising Non-Viable Streptococcus pyogenes for Injection
Streptococcus pyogenes (A Group, Type 3, Su strain) are cultured in appropriate culture media. After appropriate cultivation period the bacteria are collected by centrifugation, washed, resuspended, and treated with hydrogen peroxide to kill the bacteria. The killed bacteria are centrifuged, washed, and resuspended in suspension medium, such as Berheimers basal medium (BBM) and filtered. The bacterial suspension is treated with benzylpenicillin and heated at 37° C. for ˜10-45 min and at 45° C. for ˜20-60 min. The final bulk suspension is prepared. Vials are filled with the final bulk suspension and lyophilized. The quantitative formulae for different proposed dosage strengths of an exemplary composition comprising non-viable Streptococcus pyogenes are presented in Table 16. These compositions are based on the lyophilized product. All vial strengths are filled with the same volume (0.41 mL) of suspension before lyophilization.







TABLE 16







Quantitative Formula for Different Dosage Strengths for Exemplary Composition









Content per Vial at Different Dosage Strengths














0.02 mg
0.05 mg
0.1 mg
0.3 mg
0.5 mg
0.7 mg


Composition
(0.2 KE)
(0.5 KE)
(1 KE)
(3 KE)
(5 KE)
(7 KE)
















Non-viable
 0.02 mg
 0.05 mg
 0.1 mg
 0.3 mg
 0.5 mg
 0.7 mg


Streptococcus
 (0.21%)
 (0.49%)
 (0.88%)
 (1.93%)
 (2.54%)
 (2.93%)


pyogenes








(dried cell








mass basis)








Maltose
 9.10 mg
 8.90 mg
 8.58 mg
 7.28 mg
 5.99 mg
 4.69 mg



(94.31%)
(86.65%)
(75.77%)
(46.95%)
(30.39%)
(19.63%)


Magnesium 
 0.02 mg
 0.05 mg
 0.10 mg
 0.29 mg
 0.49 mg
 0.68 mg


sulfate
 (0.20%)
 (0.48%)
 (0.86%)
 (1.89%)
 (2.48%)
 (2.86%)


Potassium 
 0.10 mg
 0.24 mg
 0.49 mg
 1.46 mg
 2.44 mg
 3.42 mg


dihydrogen
 (1.01%)
 (2.38%)
 (4.31%)
 (9.44%)
(12.39%)
(14.30%)


phosphate








Sodium 
0.006 mg
0.015 mg
 0.03 mg
 0.09 mg
 0.15 mg
 0.21 mg


chloride 0.9%
 (0.06%)
 (0.15%)
 (0.26%)
 (0.58%)
 (0.76%)
 (0.88%)


(normal saline)








Methionine
 0.04 mg
 0.10 mg
 0.20 mg
 0.61 mg
 1.02 mg
 1.43 mg



 (0.42%)
 (1.00%)
 (1.81%)
 (3.96%)
 (5.20%)
 (6.00%)


Penicillin G 
 0.36 mg
 0.91 mg
 1.82 mg
 5.47 mg
 9.11 mg
12.75 mg


Potassium
 (3.78%)
 (8.87%)
(16.09%)
(35.24%)
(46.24%)
(53.39%)


(Benzylpenicillin)a





aBased on 1667 penicillin units/mg


KE = klinische einheit, defined as clinical unit, corresponding to 0.1 mg non-viable Streptococcus pyogenes dried cell mass






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







TABLE 17







Example of Exemplary Composition Excipients After Suspension 


with 0.9% Saline












Quantity 
0.2 mg 




per
Suspension 



Quantity 
0.2 mg 
in 0.9%



per viala
Dose
saline at 0.01


Component
(mg)
(mg)
mg/mL (% w/v)













Non-viable Streptococcus
0.1
0.2
0.002%


pyogenes (dried cell mass)





Benzyl penicillin potassiumb
1.82 mg 
3.64 mg 
0.036%


(Penicillin G potassium)
(3034
(6068




units)
units)



Methionine
0.20
0.40
0.004%


Maltose
8.58
17.16
 0.17%


Magnesium sulfate
0.10
0.20
0.002%


Potassium dihydrogen
0.49
0.98
 0.01%


phosphatec





Sodium Chloride
0.03
0.06
 0.90%


Water
NA
NA
q.s. to 100%





aBased on a 0.1 mg (1 KE) vial


bFor penicillin, there are 1667 units per mg


cThe IID name is monobasic potassium phosphate


KE = klinische einheit, defined as clinical unit, corresponding to 0.1 mg non-viable Streptococcus pyogenes;


NA = not applicable;


q.s. = quantity sufficient;






For dosing, the lyophilized powder is suspended in an isotonic sodium chloride solution to prepare a suspension at a concentration of 0.005-0.01 mg/mL. The volume of product delivered to a subject at this concentration may vary.
Example 7: In Vitro and In Vivo Efficacy Non-Viable Cells of Streptococcus pyogenes in Monotherapy and in Combination with Immune Checkpoint Inhibitors
Materials and Methods
Immunogenic Cell Death
To evaluate release of damage associated molecular patterns molecules (DAMPs), Bladder tumor MB49 cells were seeded in 96 well plate (2×104 cells/well) in triplicate for each experimental point and incubated in DMEM High Glucose phenol red-free medium with HEPES (Thermostat, Cat. No. 21063029), 10% Heat inactivated Fetal bovine Serum (HI FBS) (Seradigm Avantor, Cat. No. 1500-500H), and 1% of Penicillin/Streptomycin (P/S) (Gibco-Thermofisher, Cat. No. 15140-122), for 24 h at 37° C., 5% CO2.
Cells were treated with Composition-002 at 0, 0.2, 0.8, 3.2 and 12.8 KE/mL (1 KE=0.1 mg) for 24 hr or 1 μM of Mitoxantrone (Sigma, Cat. No. M6545) as a positive control. After treatment, the plate was centrifuged for 5 min at 400 g and supernatant was collected for HMGB1 quantification while cells were collected for Flow Cytometry analysis.
HMGB1 was quantified using Promega Lumit Immunoassay (Promega, Cat. No. W6110) kit according to the manufacturer's instructions. The average value of the appropriate background control RLU (media only, or media only with drug treatment at the corresponding concentration) was subtracted from each triplicate of treated cell sample data. The fold induction of treated samples was calculated according to the following equation: treated cells RLU−cell−free media+drug RLU)/(average of untreated cells RLU−cell−free media RLU).
Annexin V-FITC (Abcam, Cat. No. Ab14085) and Calreticulin-AF700 (R&D Systems, Cat. No. IC38981N) markers were quantified by Flow Cytometry. Briefly cells were stained with calreticulin-AF700 for 30 min at 4° C., washed and resuspended with 1× binding buffer from Annexin V kit (Abcam, Cat. No. Ab14085). Annexin V and 50 μg/mL of propidium iodide (Abcam Cat. No. Ab14085) were added in the solution and incubated for 5 min 25° C. in the dark.
For external ATP analysis (eATP), RealTimeGlo eATP Assay Reagent (Promega, Cat. No. GA5010) was added to the medium prior to COMPOSITION-002 treatment and eATP luminescent measurement was taken after 24 h of treatment. After final measurement was taken, 400 μg/mL of Digitonin (Promega, Cat. No. G9441) was added to the media to assess total ATP and general cell health as assay control. eATP was calculated using the formula [(average of triplicate cellstreated RLU−average of cell−free media+drug RLU)/(average of cells untreated RLU−cell−free media RLU)×100−100], where RLU represents Background-subtracted luminescence.
Dendritic Cell Activation and Phagocytosis Assay
Bone marrow cells were collected by flushing femurs with RPMI 1640 media (ATCC, Cat. No. 30-2001). Disaggregate cells were filtered through a 70-μM pre-wetted filter twice to remove cell clumps and counted with 3% acetic acid with methylene blue (StemCell Technologies, Cat. No. 07060). Bone Marrow cells were then resuspended in RPMI 1640 media supplemented with 2 mM GlutaMax (ThermoFisher, Cat. No. 30-2001), 10% HI FBS (Avantor, Cat. No. 1500-500H), 1% P/S (Gibco, Cat. No. 15140-122), 50 ng/ml GM-CSF (PeproTech, Cat. No. 300-03), 25 ng/mL of IL-4 (PrepoTech, Cat. No. 200-04) and cultured in 96 well plate (3×104 cells/well) at 37° C. with 5% CO2 for 48 h. Dendritic cells (DCs) differentiation continued after refreshing half of the media for 24 h. Next, cell media was completely refreshed, and cells were cultured for another 72 h to complete DCs differentiation.
MB49 cells (2.5×106 cells) were cultured in DMEM High Glucose medium with HEPES (ThermoFisher, Cat. No. 12430054), 10% HI FBS, and 1% P/S in T-25 flasks and treated with COMPOSITION-002 (0, 0.05, 0.2, 0.8, 1.6, and 3.2 KE/mL) for 24 h. Dinaciclib (1 μM) (Tocris R&D, Cat. No. 7336) was used as positive control. After treatment, cells were resuspended, washed to remove COMPOSITION-002, and stained with Vybrant™ DiO Cell-Labeling Solution (ThermoFisher, Cat. No. V22886) for 20 min at 37° C.
Pre-labeled MB49 cells and DCs were co-cultured in 96 well plates at 2:1 ratio (3×104 DCs: 1.5×104 MB49 cells) for 24 h using DC medium: MB49 medium ratio at 1:1. CD80-PE (BioLegend, Cat. No. 305207), CD86-BV421 (BioLegend, Cat. No. 305425), CD11c-AP (BioLegend, Cat. No. 337207), HLA-DR-BUV395 (BD Bioscience, Cat. No. 565972) were used to identify DCs, DiO-FITC was used to identify pre-labeled MB49 cells, and overall cell viability was analyzed by APC-eFluor780 (eBioscience, Cat. No. 65-0865-14) by Flow Cytometry.
COMPOSITION-002 Cytotoxicity and Cytokine Release
Bladder cancer RT112 cells were cultured in EMEM media (ATCC, Cat. No. 30-2003) supplemented with 2 mM GlutaMax (ThermoFisher, Cat. No. 35050061), 1% NEAA (ThermoFisher, Cat. No. 11140050), 10% HI FBS and 1% P/S. 5637 cells were cultured in RPMI 1640 media (ATCC, Cat. No. 30-2001) supplemented with 2 mM Glutamax, 10% HI FBS and 1% P/S. Both cell lines were pre-labeled using CellTracker Red solution (1 uM) (Invitrogen, Cat. No. C34552) for 30 min at 37° C. Next, cells were washed and plated in 96 well plate (2.5×104 cells/well cell/well) for 24 h at 37° C. 5% CO2.
Fresh Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using EasySep Direct Human PBMC isolation kit (Stem Cell Technologies, Cat. No. 19654) according to manufacturer's instructions and supplemented with 6 mM of EDTA (ThermoFisher, Cat. No. 15575-038). PBMC were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P/S.
COMPOSITION-002 treatment (0.2 KE/mL) was carried out in the context of tumor cells alone (2.5×104 cells/well) or in co-culture with PBMC (1.65×105 cells/well) using a ratio of 6.6:1 effector: target cells in the presence of 1.25 μg/mL Anti-CD3 (eBioscience, Cat No. 16-0037) for 72 h in 37° C., 5% CO2. After treatment, the plate was spun at 400 g for 5 min and supernatants were collected. Inflammatory cytokines were stained using the V-Plex Proinflammatory Panel 1 Human (Meso Scale, Cat. No. K15049 D-1) and detected using a plate reader. Next, cells were washed and trypsinized for Flow Cytometry analysis. CellTracker Red-PE (Invitrogen, Cat. No. C34552), Live/Dead Dye-efluor780 (eBioscience, Cat. No. 65-0865-14) and CD45-BUV395 (BD, Cat. No. 563792) were used to quantify tumor cells viability and exclude PBMCs from the analysis.
T Cell Activation
Frozen PBMC were thaw and cells were gently disassociated by resuspending them in RPMI-1640 Medium supplemented with 10% HI FBS and 1% P/S with 100 ug/mL of DNase I (StemCell Technologies, Cat. No. 17951) and incubating for 15 min at 25° C. EasySep buffer (StemCell Technologies, Cat. No. 20144) was added to the mixture and cells were filtered through 37 μM cell strainer (StemCell Technologies, Cat. No. 07900). T cells were isolated using EasySep Human T Cell isolation Kit (StemCell, Cat. No. 17951) according to the manufacturer's instructions.
Cells were plated in 96-well plate (5×105/well) and treated with COMPOSITION-002 (0.2 KE/mL) for 72 h in 37° C., 5% CO2. Plate was spun and supernatant was collected for IFN-g (Invitrogen, Cat. No. BMS228) and Granzyme B (Invitrogen, Cat. No. BMS2027-2) quantification by ELISA. T cells were collected and stained for the following markers: CD4-BUV395 (BD, Cat. No. 564724), FoxP3-PE (BD, Cat. No. 560852); CD8-FITC (BioLegend Cat. No. 344704), PD-1-PE-Cy7 (BioLegend, Cat. No. 329918), CTLA-4-BV421 (BioLegend, Cat. No. 369606), TIGIT-BV711 (BioLegend Cat. No. 372742), TIM3-BV605 (BioLegend Cat. No. 345018), LAG3-APC (BioLegend Cat. No. 369212), Ki67-efluor506 (eBioscience Cat. No. 69-5698-82), Live/Dead dye-efluor708 (eBioscience, Cat. No. 65-0865-14). Data were acquired by Flow Cytometry.
PD-L1 Analysis in Tumor Cells
Bladder cancer 5637 cells cultured in RPMI-1640 media supplemented with 2 mM Glutamax, 10% HI FBS and 1% P/S are pre-labelled with Cell Tracker Red and plated in 96-well plate 2.5×104cells/well) to grow overnight.
Frozen PBMC were thaw and cells were gently disassociated by resuspending them in RPMI-1640 Medium supplemented with 10% HI FBS and 1% P/S with 100 μg/mL of DNase I and incubating for 15 min at 25° C. EasySep buffer was added to the mixture and cells were filtered through 37 μM cell strainer. T cells were isolated using EasySep Human T Cell isolation Kit (StemCell, Cat. No. 17951) according to the manufacturer's instructions and kept in RPMI-1640 Medium supplemented with 10% HI FBS and 1% P/S.
T cells were added to appropriate co-culture wells following the 6.6:1 ratio (1.65×105 cells cell/well) with media ratio for 5637:T cells of 1:1. 5637 cells alone and in co-culture with T cells were treated with COMPOSITION-002 (0.2 and 0.8 KE/mL) for 72 h at 37° C., 5% CO2. After treatment, supernatant was washed and PD-L1 (BioLegend, Cat. No. 374510) marker was analyzed in pre-labeled tumor cells by flow cytometry.
In Vitro Cytotoxicity Analysis Using xCELLigence Real-Time Cell Analyzer (RTCA)
Fresh Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using 2× EasySep Direct Human PBMC isolation kit (Stem Cell Technologies, Cat. No. 19654) according to manufacturer's instructions and supplemented with 6 mM of EDTA (ThermoFisher, Cat. No. 15575-038). PBMC were resuspended in RPMI-1640 supplemented with 10% HI FBS and 1% P/S. 5637 cells cultured in RPMI-1640 media supplemented with 10% HI FBS and 1% P/S are seeded in 96-well E-Plate (Agilent, Cat. No. 300600910) (5×105) cells per well) for 30 min for the cells to adhere. After ˜78 hours, the effector cells (human PBMCs; effector/target ratio 6.6:1) and treatments were added. COMPOSITION-002 treatment (0.8 KE/mL) was carried out for −65 hours in the co-culture setting (Tumor cells+PBMC) alone or in combination with the following antibodies: Anti-PD-1 (10 μg/mL-Bioxcell, Cat. No. SIM0010), Anti-PD-L1 (10 μg/mL-Bioxcell, Cat. No. SIM0009), Anti-CTLA-4 (10 μg/mL-Selleckchem, Cat. No. A20001). RecombiMAb IgG4 (Bioxcell, Cat. No. CP147) (10 μg/mL), and RecombiMAb IgG1 (Bioxcell, Cat. No. CP147) (10 μg/mL) were used as isotype controls. Cell Index measurements were collected by xCelligence RTCA eSight (Agilent) every 15 min for a total of 143.5 hours (˜78 hours cancer cells alone+˜65 hours in co-culture). For each well, the % cytolysis was calculated utilizing the normalized sample cell index and the normalized average target alone control according to the following equation:



 
  
   %
   ⁢
      
   Cytolysis
  
  =
  
   
    
     (
     
      
       Cell
       ⁢
          
       
        Index
        
         no
         ⁢
            
         effector
        
       
      
      -
      
       Cell
       ⁢
          
       
        Index
        effector
       
      
     
     )
    
    
     Cell
     ⁢
        
     
      Index
      
       no
       ⁢
          
       effector
      
     
    
   
   ×
   100
  
 



MB49 Subcutaneous Mouse Model

For the dose finding study, 14-week-old Female C57BL/6 mice were implanted subcutaneously with MB49 bladder cancer cells (2.0×105 cells/mouse) and 10 mice per group were randomized after 8 days (80-130 mm3 tumor size for enrolment). Mice were dosed individually once a week for 4 weeks with COMPOSITION-002 intravenously (0.08, 0.4 and 2 KE/mouse). Tumor measurements were done twice a week using calipers. Animals that did not reach humane endpoint (weight loss >20%, tumor burden >2000 mm3, open weeping tumor ulceration, severe respiratory distress, severely impaired movement, or loss of righting reflex) were monitored for a maximum of 35 days.
For combination study, 14 weeks old female mice were implanted subcutaneously with MB49 cells (2.0×105 cells/mouse) and 10 mice per group were randomized after 6 days (75-130 mm3 of tumor size for enrolment). Mice were dosed individually once a week for 4 weeks (0.4 KE/mouse of COMPOSITION-002) intravenously and/or anti-PD-1 twice a week for 2 weeks (10 mg/Kg) (BioXcell, Cat No. BP0146). Isotype Control (2A3) were used as negative control (BioxCell, Cat. No. BP0089). Animals that did not reach humane endpoint were kept for 5 days after the last dose of COMPOSITION-002 before being euthanized and have tumors collected and processed for immunohistochemistry.
EMT6 Orthotopic Mouse Model.
For the dose finding study, 14-weeks old female Balb/c mice were implanted with 5×105 EMT6 triple negative breast cancer cells on their left 4th mammary fat pad. After 7 days, six mice were randomized per group (50-150 mm3 of tumor size for enrollment) and were dosed with COMPOSITION-002 twice a week (0.4, 1 and 2 KE/mouse) intravenously. Tumor growth and mice body weight were measured twice a week for 3 weeks. Mice that did not reach humane endpoint were euthanized on day 35 of the study. Tumors of 3 mice from each group were collected and after mechanical dissociation, were treated with collagenase D (2.5 mg/mL) for chemical separation. Cells were filtered through 70 μm cell strainer and stained for FACS analysis of tumor infiltrated immune cell populations (Table 18).







TABLE 18







FACS Analysis of Tumor Infiltrated Immune Cell Populations








Immune Cell Population
Phenotypic Markers





T cells
CD45+CD3+


CD4+ T cells
CD45+CD3+CD4+


CD8+ T cells
CD45+CD3+CD8+


CD8+ T cells
CD45+CD3+CD4−CD8+


Tregs
CD3+CD4+Foxp3+CD25+


Granulocytic MDSC
CD45+CD3 CD11b+Ly6G+Ly6Clow


Monocytic MDSC
CD45+CD3 CD11b+Ly6G−Ly6Chigh


NK cells
CD45+CD3−CD49b+CD335+


M1 macrophages
CD45+CD3−F4/80+CD206−


M2 macrophages
CD45+CD3−F4/80+CD206+


PD1 positive
PD1+









Results:

MB49 bladder cancer exposed to COMPOSITION-002 undergo apoptosis (FIG. 28A) with production of hallmarks of immunogenic cell death such as HMGB1, extracellular ATP (eATP) and expression Calreticulin on the cell surface (FIGS. 28B-28D). These damage-associated molecular patterns (DAMPS) serve as signals to attract and activate antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs) which in turn can effectively activate naïve T cells. As DCs play the key role in the recognition of DAMPs associated with immunogenic cell death and the subsequent uptake and presentation of tumor antigens, the phagocytosis of COMPOSITION-002-treated tumor cells by DCs was examined. MB49 cells were treated with COMPOSITION-002 and then cultured with mouse bone marrow-derived DCs. COMPOSITION-002-treated MB49 bladder cancer cells increased dendritic cell phagocytosis (FIG. 29A) and phenotypic maturation, as indicated by the upregulated surface expression of CD80, CD86, and HLA-DR increased (FIGS. 291B-291D). Using an in vitro co-culture approach of bladder cancer cells (5637 and RT112) and PBMCs, COMPOSITION-002 was found to enhance immune-mediated bladder cancer cell killing (FIGS. 30A-30B). Of note, cytokine analyses showed that COMPOSITION-002 increased the release of pro-inflammatory Th1 cytokines (FIG. 31A) creating a favorable environment for the induction of cellular and humoral anti-tumor immunity. Additionally, COMPOSITION-002 also promoted reduction of Th2 cytokines (FIG. 31B), which are correlated with tumor growth. For example, COMPOSITION-002 reduced IL-10 release, which is known to inhibit the secretion of various Th1 cytokines by macrophages and dendritic cells.
To investigate if COMPOSITION-002 induces PD-L1 expression in cancer cells, thereby suppressing the antitumor immune response, bladder cancer cells (5637), in coculture with PBMCs, were treated with increasing concentration of COMPOSITION-002. COMPOSITION-002 treatment led to increased PD-L1 expression in 5637 cells in comparison to the control (FIG. 34). Moreover, COMPOSITION-002 stimulation of T-cells alone increased T cell proliferation as observed by high percentage of CD4 and CD8 cells exhibiting KI67 marker (FIGS. 32A and 32H). Additionally, COMPOSITION-002 promoted IFN-γ and Granzyme B release (FIGS. 33A-33B) suggesting enhanced cytotoxic activity. However, markers of exhausted T-cell phenotypes were identified through high levels of LAG3, CTLA4, PD-1, TIGIT, and TIM3 in CD4 and CD8 T-cells (FIGS. 32B 32C, 32D, 32E, 32F, 32J, 32I, 32K, 32L, and 32M, respectively) as well as FOXP3 CD4+ regulatory T-cells (FIG. 32G). Taken together, the immunostimulatory action of COMPOSITION-002 may be potentiated with additional agents targeting these exhausted T-cell phenotypes.
To investigate the potential synergic antitumor effect of COMPOSITION-002 with the immune checkpoint inhibitors (ICIs) anti-PD-L1 antibody, anti-CTLA4 antibody, and anti-PD1 antibody, in vitro, the xCELLigence platform was selected, which can be used monitoring cell health, proliferation, and cytolysis over time. Target 5637 bladder cancer cells were seeded in the biosensor plate (E-Plate) and allowed to attach and proliferate. After 78 h, fresh PBMCs were added on top of the 5637 cells in the presence or absence of COMPOSITION-002, ICIs or irrelevant IgGs isotypes (IgG4 for anti-PD1 and IgG1 for anti-PD-L1 and anti-CTLA4) used as controls for ˜65 hours (143.5 hours total). The combination of COMPOSITION-002 with anti-PD-L1 or anti-CTLA4 showed enhancement of 5637 cell cytolysis in the presence of human PBMCs when compared to individual treatments (FIGS. 35A-35D). It is worthwhile to mention that the combination of COMPOSITION-002 and ICIs had a delayed anti-tumor effect compared to COMPOSITION-002 alone, suggesting that the combination activity is dependent on the COMPOSITION-002 mediated-expression of immune checkpoint (e.g., PD-L1, CTLA4). However, just a slight increase of cytolysis was observed when anti-PD1 antibody was used when compared to COMPOSITION-002 alone (FIGS. 35E-35F).
As the response of tumors to immunotherapy, such as anti-PD-1, may depend on interactions of several cell types in the tumor microenvironment in vivo, the COMPOSITION-002 and anti-PD1 combination study was repeated using a bladder cancer mouse model. Instead of the MBT2 model previously used (Example 2), the MB49 murine bladder carcinoma model was selected, which has the potential to respond to immune stimulants and is widely used in bladder cancer immunotherapy research. Moreover, based on the observed delayed anti-tumor effect of COMPOSITION-002 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). COMPOSITION-002, as a monotherapy, was efficient in reducing tumor growth and increasing survival in a MB49 subcutaneous model (FIGS. 36A-36B). Further analysis using COMPOSITION-002 in combination with anti-PD-1 in this same model revealed reduction in tumor volume compared to either COMPOSITION-002 or anti-PD-1 as monotherapy (FIGS. 37A-37E). For example, the mean tumor volume for anti-PD1 alone and for the combination group on Day 20 was 264 mm3 and 159 mm3, respectively, which indicates additional efficacy. Furthermore, 30% of complete regressions and 10% tumor free survivors were observed in the combination treatment group. No complete regression nor tumor free survivors were found in the monotherapy groups.
The antitumor 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-PD1 treatment. Results demonstrated a significant but modest anti-tumor effect by COMPOSITION-002 administered intravenously as monotherapy (FIG. 38A). On immune cells analyzed by flow cytometry, tumor associated macrophages (TAMs) were identified, with an increase of the anti-tumor macrophages 1 (M1) and decrease of pro-tumor macrophages 2 (M2) cell types (i.e., M1 polarization), which were associated with increased PD-1 expression on T cells (FIG. 38B). Systemic delivery of COMPOSITION-002 in combination with anti-PD1 in the EMT6 TNBC mouse model demonstrated significantly superior anti-tumor activity compared to each agent as monotherapy, indicating synergism between COMPOSITION-002 and anti-PD1 (FIGS. 39A-39B).
Furthermore, the combination treatment of COMPOSITION-002 with anti-PD1 re-shaped the local immune cell populations by restoring PD-1 levels in T cells, increasing levels of intratumor CD4 T-cells, promoting macrophage polarization towards M1 phenotype (anti-tumor), downregulating pro-tumor Treg cells (FIGS. 40A-40C). The fact that COMPOSITION-002 in combination with anti-PD1 significantly increases the level of tumor-infiltrating Natural Killers cells (NK) compared to monotherapy groups (FIG. 40D) is particularly important. Indeed, researchers have highlighted the advantages of anti-PD-1/PD-L1 therapy in improving NK cell function and found that interrupting the PD-1/PD-L1 interaction can enhance the killing effect of NK cells on tumor cells. Moreover, PD-1/PD-L1 antibodies are completely ineffective in some NK-deficient mouse models.
Representative Embodiments
1. A method 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.
2. The method of embodiment 1, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered intratumorally, intravenously, intramuscularly, subcutaneously or intraperitoneally.
3. The method of embodiment 1 or 2, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of about 10 KE or greater.
4. The method of embodiment 3, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of at least 20 KE.
5. The method of embodiment 3, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of at least 40 KE.
6. The method of any one of embodiments 1-5, wherein the non-viable cells of Streptococcus pyogenes is 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, KE, 40 KE, 50 KE, 60 KE, 70 KE, 80 KE, 90 KE, or 100 KE.
7. The method of any one of embodiments 1-6, wherein the non-viable cells of Streptococcus pyogenes comprise cells of the Su strain of Streptococcus pyogenes. 
8. The method of any one of embodiments 1-7, 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 embodiments 1-8, wherein the immune checkpoint inhibitor is an inhibitor of PD1/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 embodiment 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 embodiment 10, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrelimab, cemiplimab, sasanlimab, nofazinlimab, geptanolimab, zimberelimab, serplulimab, pucotenlimab, prolgolimab, camrelizumab, cadonilimab, dorstarlimab, penpulimab, toripalimab, tislelizumab, sintilimab, or dostarlimab.
12. The method of embodiment 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 of embodiment 12, wherein the PD-L1 antibody comprises atezolizumab, durvalumab, envafolimab, sugemalimab, cosibelimab, socazolimab, tagitanlimab, betifisolimab, lesabelimab, pacmilimab, or avelumab
14. The method of embodiment 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 embodiment 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 embodiment 9, wherein the immune checkpoint inhibitor comprises a CTLA-4 inhibitor, optionally wherein the CTLA-4 inhibitor is an antibody.
17. The method of embodiment 16, wherein the CTLA-4 antibody comprises ipilimumab, tremelimumab, tuvonralimab.
18. The method of any one of embodiments 1-17, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered at least one day prior to the immune checkpoint inhibitor.
19. The method of any one of embodiments 1-18, further comprising administering an additional anti-cancer agent to the subject.
20. The method of any one of embodiments 1-19, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered prior to, concurrently with, or subsequent to the immune checkpoint inhibitor.
21. The method of any one of embodiments 1-20, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [A Group, Type 3] Su strain.
22. The method of any one of embodiments 1-21, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes. 
23. The method of any one of embodiments 1-22, 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 of any one of embodiments 1-23, 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 embodiments 1-24, wherein the triple negative breast cancer is metastatic.
26. The method of any one of embodiments 1-25, wherein the triple negative breast cancer is recurrent.
27. The method of any one of embodiments 1-26, wherein the triple negative breast cancer is completely or partially resistant to a PD-1 inhibitor, 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 for treating triple negative breast cancer.
29. A medicament comprising non-viable cells of Streptococcus pyogenes for use in combination with an immune checkpoint inhibitor for treating 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 embodiment 30, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered intravesically, intratumorally, intravenously, intramuscularly, subcutaneously, or intraperitoneally.
32. The method of embodiment 30 or 31, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of about 10 KE or greater.
33. The method of embodiment 32, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of at least 20 KE.
34. The method of embodiment 32, wherein the non-viable cells of Streptococcus pyogenes is present in the composition in the amount of at least 40 KE.
35. The method of any one of embodiments 30-34, wherein the non-viable cells of Streptococcus pyogenes is 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, KE, 40 KE, 50 KE, 60 KE, 70 KE, 80 KE, 90 KE, or 100 KE.
36. The method of any one of embodiments 30-35, wherein the non-viable cells of Streptococcus pyogenes comprise cells of the Su strain of Streptococcus pyogenes. 
37. The method of any one of embodiments 30-36, 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 embodiments 30-37, wherein the immune checkpoint inhibitor is an inhibitor of 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 embodiment 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 embodiment 39, wherein the PD-1 antibody comprises pembrolizumab, nivolumab, cetrelimab, cemiplimab, sasanlimab, tislelizumab, or dostarlimab.
41. The method of embodiment 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 embodiment 41, wherein the PD-L1 antibody comprises atezolizumab, durvalumab, envafolimab, or avelumab
43. The method of embodiment 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 embodiment 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 embodiments 30-44, wherein the non-muscle invasive bladder cancer is completely or partially resistant to the PD-1 inhibitor and/or PD-L1 inhibitor.
46. The method of embodiment 38, wherein the immune checkpoint inhibitor comprises a CTLA-4 inhibitor, optionally wherein the CTLA-4 inhibitor is an antibody.
47. The method of embodiment 46, wherein the CTLA-4 antibody comprises ipilimumab, tremelimumab, tuvonralimab.
48. The method of any one of embodiments 30-47, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered at least one day prior to the immune checkpoint inhibitor.
49. The method of any one of embodiments 30-48, further comprising administering an additional anti-cancer agent to the subject.
50. The method of any one of embodiments 30-49, wherein the composition comprising non-viable cells of Streptococcus pyogenes is administered prior to, concurrently with, or subsequent to the immune checkpoint inhibitor.
51. The method of any one of embodiments 30-50, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises Streptococcus pyogenes [A Group, Type 3] Su strain.
52. The method of any one of embodiments 30-51, wherein the composition comprising non-viable cells of Streptococcus pyogenes comprises benzylpenicillin-treated Streptococcus pyogenes. 
53. The method of any one of embodiments 30-52, 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 embodiments 30-53, 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 embodiments 30-54, wherein the subject has low grade non-muscle invasive bladder cancer.
56. The method of any one of embodiments 30-55, wherein the subject has high-grade non-muscle invasive bladder cancer.
57. The method of any one of embodiments 30-54, wherein the non-muscle invasive bladder cancer has been identified as low risk.
58. The method of any one of embodiments 30-54, wherein the non-muscle invasive bladder cancer has been identified as intermediate risk.
59. The method of any one of embodiments 30-54, wherein the non-muscle invasive bladder cancer has been identified as high risk.
60. The method of any one of embodiments 30-54, wherein the non-muscle invasive bladder cancer has been identified as high-grade Ta or T1.
61. The method of any one of embodiments 30-54, wherein the non-muscle invasive bladder cancer has been identified as cancer in situ (CIS) with or without Ta and/or T1.
62. The method of any one of embodiments 30-61, wherein the non-muscle invasive bladder cancer is recurrent.
63. The method of any one of embodiments 30-62, wherein the subject has not received prior BCG treatment.
64. The method of any one of embodiments 30-62, wherein the subject has received adequate BCG treatment.
65. The method of any one of embodiments 30-62, wherein the subject is unresponsive to BCG treatment.
66. The method of any one of embodiments 30-62, wherein the non-muscle invasive bladder cancer is BCG failed, BCG refractory, or BCG relapsing.
67. A pharmaceutical composition comprising non-viable cells of Streptococcus pyogenes for use in combination with an immune checkpoint inhibitor for treating non-muscle invasive bladder cancer.
68. A medicament comprising non-viable cells of Streptococcus pyogenes for use in combination with an immune checkpoint inhibitor for treating non-muscle invasive bladder cancer.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to 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 Jan. 9, 2023, U.S. Provisional Patent Application No. 63/487,224, filed on Feb. 27, 2023, and U.S. Provisional Patent Application No. 63/487,232, filed on Feb. 27, 2023, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.
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 the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.