Methods of treating cancer using microbe-derived vaccines

Abstract

Disclosed herein are methods of treating cancer comprising: systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof. Also disclosed herein are methods of reducing the progression of a cancer comprising: systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof. Also disclosed herein are methods of increasing the amount of resident memory T-cells (Trms) in a tumor of a subject identified as having a cancer, the method comprising systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor, wherein the amount of Trms is compared to a reference level of Trms in a tumor of the same cancer. Treated cancers can include breast and pancreatic cancers.

Description

METHODS OF TREATING CANCER USING MICROBE-DERIVED VACCINESRelated Applications

[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 472,740, filed June 13, 2023, the contents of which is hereby incorporated by reference.Technical Field

[0002] The present disclosure relates to methods for treating cancer and to methods for increasing the level of resident memory T-cells in a tumor, and in particular using microbe-derived vaccines for treating cancer and for increasing the level of resident memory T-cells in a tumor.Background

[0003] The tumor microenvironment represents a significant barrier that restricts immune responses against tumors and limits the efficacy of currently available immunotherapies as treatments for cancer. However, infiltration of tumors by immune cells has been shown to correlate with augmented responses to immunotherapy and improved survival. An immunologically inflamed (“hot”) tumor microenvironment exhibits robust antigen presentation and T-cell activation, contributing to the development of tumor-specific CD8+T cell functionality that may kill cancer cells, generate systemic tumor-specific immunity, and form long-term anti-tumor memory responses. See, for example, M. Ayers et al., The Journal of clinical investigation 127, 2930-2940 (2017) and Martfnez-Lostao et al., Clinical Cancer Research 21 , 5047-5056 (2015). However, a significant proportion of patients harbor a immunologically non-responsive (“cold”) tumor microenvironment that may either be devoid of immune cell infiltration (an “immune desert”) or that is predominantly infiltrated by suppressive regulatory cell subtypes (including regulatory T-cells [Tregs], regulatory B cells [Bregs], and myeloid-derived suppressor cells [MDSCs]). See, for example, Kather et al., eLife 7 (2018), Lindau et al.,Immunology 138, 105-115 (2013), and Mauri et al., The Journal of Clinical Investigation 127, 772-779 (2017). In both immune deserts and suppressive cell-type dominated environments, cancer growth may be immunologically unchecked. Therefore, to increase response rates to immunotherapy, innovative solutions are needed to convert “cold” tumor microenvironments to “hot” tumor microenvironments.

[0004] One proposed strategy to initiate a robust immune response in immunologically cold tumors is the introduction of “non-self” antigens, including microbe-derived antigens. Recently, Listeria monocytogenes was engineered to deliver tetanus toxoid (TT) in pancreatic ductal adenocarcinoma (PDAC) tumors and demonstrated robust and durable anti-cancer effects in mice. See, for example, Selvanesan et al. Sci Transl Med 14, eabc1600 (2022). However, the usage of live bacteria, even attenuated strains, may be highly precarious and lead to adverse toxicities. See, for example, Gupta et al. Vaccines (Basel) 9 (2021 ). Thus, there is a need for additional microbe-derived antigens to induce immune responses in tumors.Summary

[0005] In the present disclosure, it is shown that microbe-derived vaccines, and their components, may augment an anti-tumor immune response within the tumor microenvironment, ultimately converting immunologically “cold” tumors to “hot” by increasing the number of resident memory T-cells (Trm) found in the tumor. This results in reduced local tumor growth, limited cancer progression, and decreased metastases. Furthermore, it has been demonstrated herein that such outcomes can be achieved by intraperitoneal administration of vaccines.

[0006] Disclosed herein are methods of treating cancers. The methods include systemically administering a therapeutically effective dose of one or more microbe- derived vaccines to a subject in need thereof. The methods may also include intraperitoneally administering a therapeutically effective dose of one or more microbe- derived vaccines to a subject in need thereof.

[0007] Also disclosed herein are methods of reducing the progression of a cancer. The methods include systemically administering a therapeutically effective dose of oneor more microbe-derived vaccines to a subject in need thereof. The methods may also include intraperitoneally administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof.

[0008] Also disclosed herein are methods of increasing the amount of resident memory T-cells (Trms) in a tumor of a subject identified as having a cancer. The methods include the systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor, where the amount of Trms is compared to a reference level of Trms in a tumor of the same cancer. The methods may also include the intraperitoneally administering a therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor, where the amount of Trms is compared to a reference level of Trms in a tumor of the same cancer.

[0009] In any of the methods disclosed herein, the cancer is pancreatic or breast cancer. In some cases, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In some cases, the breast cancer is triple negative breast cancer (TNBC). In some cases, the one or more microbe-derived vaccines is a meningococcal vaccine, a Haemophilus influenzae vaccine, or a pneumococcal vaccine. In some cases, the one or more microbe-derived vaccines is two or more microbe-derived vaccines. In some cases, the therapeutically effective dose includes one or more doses of the one or more microbe-derived vaccine. In some cases, at least one of the one or more doses is administered before the subject is identified as having the cancer.

[0010] In any of the methods disclosed herein, the method may also include administering at least one additional anticancer treatment. In some cases, the at least one additional anticancer treatment is surgical therapy, chemotherapy, radiation therapy, hormonal therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radioablation, or a biological therapy. In some cases, the at least one additional anticancer treatment is an immune checkpoint inhibitor. In some cases, the at least one checkpoint inhibitor is an inhibitor of Cytotoxic T-lymphocyte antigen 4 (CTLA-4).

[0011] In any of the methods disclosed herein, the method may also include identifying the subject as having the cancer.

[0012] In any of the methods disclosed herein, the method may also include determining the amount of Trms in the tumor after administering the therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor.

[0013] In any of the methods disclosed herein, the method may also include determining the reference level of Trms in a tumor of the same cancer.Brief Description of the Drawings

[0014] Figure 1A is a schematic representation of the infiltration of immune cells in tumor treated with Meningococcal conjugate vaccine (MCV) in otherwise immunological cold PDAC. Figure 1 B is a graph showing MCV administration increases TLR2, TLR4, TLR7 and TLR9 activity the HEK-Blue-mTLR cell lines expressing the indicated TLR type (TLR2, TLR4, TLR7 or TLR9) in comparison to matched parental cell lines (Null).

[0015] Figures 2A-2C is a schematic diagram (Figure 2A) for treatment schedule with MCV and a graph showing PDAC mean tumor progression measured after treatment with PBS and MCV in High and low T-cell infiltration, respectively (Figure 2B- 2C).

[0016] Figures 3A-3B are graphs showing intratumoral (IT) administration of asplenia vaccines (Meningococcal Conjugate Vaccine (Meningo or MCV), Haemophilus influenzae vaccine (H. flu), or Pneumococcal vaccine (Pneumo), administered individually) limited pancreatic tumor size and reduced pancreatic tumor progression in high T-cell infiltrations tumors (2838c) (Fig. 3A) and low t-cell infiltration tumors (6419c5) (Fig. 3B).

[0017] Figures 4A-4B are graphs showing systemic administration via i.p. injection of asplenia vaccines (Meningo, H. flu, or Pneumo, administered individually) limited pancreatic tumor size and reduced pancreatic tumor progression in high T-cell infiltrations tumors (2838c) (Fig. 4A) and low t-cell infiltration tumors (6419c5) (Fig. 4B).

[0018] Figures 5A-5B are graphs showing percentages of intratumoral CD8+T-cells of the T-cell population following i.p. injection of asplenia vaccines (Meningo, H. flu, orPneumo, administered individually) in high T-cell infiltrations tumors (2838c) (Fig. 5A) and low t-cell infiltration tumors (6419c5) (Fig. 5B).

[0019] Figures 6A-6B are graphs showing percentages of intratumoral CD4+T of the T-cell population following i.p. injection of asplenia vaccines (Meningo, H. flu, or Pneumo, administered individually) in high T-cell infiltrations tumors (2838c) (Fig. 6A) and low t-cell infiltration tumors (6419c5) (Fig. 6B).

[0020] Figures 7A-7B are graphs showing percentages of intratumoral natural killer (NK) cells of the CD45+population following i.p. injection of asplenia vaccines (Meningo, H. flu, or Pneumo, administered individually) in high T-cell infiltrations tumors (2838c) (Fig. 7A) and low t-cell infiltration tumors (6419c5) (Fig. 7B).

[0021] Figures 8A-8B are graphs showing percentages of intratumoral M-Myeloid derived suppressor cells (MDSC) of CD11b+cell population following i.p. injection of asplenia vaccines (Meningo, H, Flu, or Pneumo, administered individually) in high T-cell infiltrations tumors (2838c) (Fig. 8A) and low t-cell infiltration tumors (6419c5) (Fig. 8B). Figures 8C and 8D show the percentage CD8+T-cells that secrete Granzyme B in 2838c high T-cell infiltration tumors (Fig. 8C) and 6419c5 low T-cell infiltration tumors (Fig. 8D). Figures 8E and 8F show the percentage CD4+T-cells that secrete Granzyme B in 2838c high T-cell infiltration tumors (Fig. 8E) and 6419c5 low T-cell infiltration tumors (Fig. 8F). Figures 8G and 8H show the percentage MCHII+ dendritic cells (DCs) that are CD11 C+in 2838c high T-cell infiltration tumors (Fig. 8G) and 6419c5 low T-cell infiltration tumors (Fig. 8H). Figures 8I and 8J show the percentage the CD45+cell population that are F4 / 80+and CD11b+(Macrophage) in 2838c high T-cell infiltration tumors (Fig. 8I) and 6419c5 low T-cell infiltration tumors (Fig. 8J).

[0022] Figures 9A-9B are graphs showing bioluminescence of PDAC tumor response of KPC-Luc PDAC to H. flu (Fig. 9A) and tumor growth (Fig. 9B) 0 or 10 days after administration (tDay) of vehicle only (circle) or 50pls of H. flu (square) once for three days via i.p. injection. Tumor sizes were measured by bioluminescence imaging, averaged, and graphed ± S.E. (****, P < 0.0001 ).

[0023] Figures 10A-10C are graphs showing percentages of various intratumoral immune cells in PDAC tumors as measured by flow cytometer. The percentage of CD4+T-cell population of the T-cell population (Fig. 10A), the percentage of CD8+T-cellpopulation of the T-cell population (Fig. 10B), and the ratio of the percentages of CD8+T-cells to Treg cells (Fig. 10C) are shown. N=5 mice per grouped data averaged, and graphed ± S.E. (*, P < 0.01 , **, P < 0.001 ).

[0024] Figures 11 A-11 K illustrate the effect of MCV of H. flu vaccine in an orthotopic murine model. Fig. 11 A is a schematic showing how the treatment with microbe-derived vaccines induces resident memory T-cells in the tumor microenvironment. Fig. 11 B is a schematic of the experimental design. Mice were sacrificed on Day 11 to study the immune composition, or were allowed to live until an endpoint was reached to study survival. Treatments were administered for 3 or 5 days, as indicated. Fig. 11 C is a series of representative bioluminescent images showing the response of orthotopic KPC-Luc PDAC to MCV or H. flu. Fig. 11 D is a graph of tumor growth after mice were administered vehicle only (PBS; circle), MCV (square), or H. flu (triangle) once for five days via intraperitoneal injections. Tumor sizes were measured by bioluminescence imaging, averaged, and graphed ± S.E. (****, P < 0.0001 ). Fig. 11 E is a graph of mouse survivability until death after mice were administered vehicle only (PBS; circle), MCV (square), or H. flu (triangle) (**, p <0.001 ). Figs. 11 F-11 K are a series of graphs of the percentage of FoxP3+cells (Tregs) of the CD4+T-cell population (Fig. 11 F), the percentage of CD11b+cells (MDSCs) of the CD45+T-cell population (Fig. 11G), the percentage of F4 / 80 cells (Macrophages) of the CD45+T-cell population (Fig. 11 H), the percentage of CD4+T-cells of the T-cell population (Fig. 111), the percentage of CD8+T- cells of the T-cell population (Fig. 11 J), the percentage of NK cells of the CD45+T-cell population (Fig. 11 K), and the percentage of CD69+memory T-cells of the CD8+T-cell population (Fig. 11 L); n=5 animals per treatment group. Significance / p-values were calculated by one-way ANOVA and are indicated as follows: *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001 ; ns = not statistically significant.

[0025] Figures 12A-12I shows that intraperitoneal injections of Meningococcal Conjugate Vaccine (MCV) and Haemophilus influenzae (H. flu) treated mice in orthotropic model of PDAC. Fig. 12A is a schematic of the experimental design, including two doses before mice were challenged with pancreatic cancer tumors. Fig. 12B is a series of representative bioluminescent images showing the response of orthotopic KPC-Luc PDAC to MCV or H. flu. Fig. 12C is a graph of tumor growth aftermice were administered vehicle only (PBS; circle), MCV (square), or H. flu (triangle) once for five days via intraperitoneal injections. Tumor sizes were measured by bioluminescence imaging, averaged, and graphed ± S.E. (****, P < 0.0001 ). Fig. 12D- 121 are a series of graphs of the percentage of CD69+cells of the CD4+T-cell population (Fig. 12D), the percentage of CD44+and CD62L- (low CD62 expression) cells of the CD4+T-cell population (Fig. 12E), the percentage of Granzyme B+cells of the CD4+T-cell population (Fig. 12F), the percentage of CD69+cells of the CD8+T-cell population (Fig. 12G), the percentage of CD44+and CD62L- cells of the CD8+T-cell population (Fig. 12H), and the percentage of Granzyme B+cells of the CD8+T-cell population (Fig. 121); n=5 animals per treatment group. Significance / p-values were calculated by one-way ANOVA and are indicated as follows: *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001 ; ns = not statistically significant. Fig. 12J is a supporting figure of Fig. 12E, and Fig. 12H is a supporting figure of Fig. 12H, showing CD44+and CD62L cells.

[0026] Figures 13A-13B are graphs showing percentages of intratumoral regulatory T-cells (Tregs) of the CD4+T-cell population following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 13A) and low t-cell infiltration tumors (6419c5) (Fig. 13B).

[0027] Figures 14A-14B are graphs showing percentages of intratumoral granzyme B positive (granzyme B+) of the CD8+T-cell population, indicating intratumoral cytotoxic CD8+T-cell function, following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 14A) and low t-cell infiltration tumors (6419c5) (Fig. 14B).

[0028] Figures 15A-15B are graphs showing percentage of intratumoral granzyme B positive (granzyme B+) of the CD4+T-cell population, indicating intratumoral cytotoxic CD4+T-cell function, following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 15A) and low t-cell infiltration tumors (6419c5) (Fig. 15B).

[0029] Figures 16A-16B are graphs showing ratios of intratumoral CD8+T-cell to intratumoral Treg, indicating an anti-tumor or pro-tumor tumor microenvironment,following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 16A) and low t-cell infiltration tumors (6419c5) (Fig. 16B).

[0030] Figures 17A-17B are graphs showing increased percentage of intratumoral CTLA-4 expressing CD8+T-cells following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 17A) and low t-cell infiltration tumors (6419c5) (Fig. 17B).

[0031] Figures 18A-18B are graphs showing the percentage of intratumoral PD-1 expressing CD8+T-cells following i.p. injections of asplenia vaccines (Meningo, H. flu, or the combination of H. flu + Meningo) in high T-cell infiltrations tumors (2838c) (Fig. 18A) and low t-cell infiltration tumors (6419c5) (Fig. 18B).

[0032] Figures 19A-19C describe a schematic diagram (Figure 19A) and graphs showing PDAC mean tumor progression measured after treatment with PBS, aCTLA-4, or CTLA-4 + MCV in comparison to control treatment (PBS) following i.p. injections of Meningo with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 19B) and low t-cell infiltration tumors (6419c5)(Fig. 19C). Figures 19D and 19E are graphs showing PDAC mean tumor size (a measure of tumor progression) measured after treatment with and without anti-PD-1 ICI treatment in high T-cell infiltrations tumors (2838c) (Fig. 19D) and low t-cell infiltration tumors (6419c5)(Fig. 19E). Figures 19F and 19G are graphs showing PDAC mean tumor size (a measure of tumor progression) measured after treatment with and without anti-PD-1 and anti-CTLA- 4 ICI treatment in high T-cell infiltrations tumors (2838c) (Fig. 19F) and low t-cell infiltration tumors (6419c5)(Fig. 19G).

[0033] Figures 20A-20B are graphs showing the percentage of intratumoral CD4+T- cells of the T-cell population following i.p. injections of Meningococcal Vaccine (Meningo) with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 20A) and low t-cell infiltration tumors (6419c5) (Fig. 20B).

[0034] Figures 21 A-21 B are graphs showing the percentage of intratumoral CD8+T- cells of the T-cell population following i.p. injections of Meningococcal Vaccine (Meningo) with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 21 A) and low t-cell infiltration tumors (6419c5) (Fig. 21 B).

[0035] Figures 22A-22B are graphs showing the percentage of intratumoral natural killer (NK) cells in CD45+T-cell population following i.p. injections of Meningococcal Vaccine (Meningo) with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 22A) and low t-cell infiltration tumors (6419c5) (Fig. 22B).

[0036] Figures 23A-23B are graphs showing the percentage of intratumoral granzyme B positive (granzyme B+) of the CD4+T-cell population, indicating enhanced intratumoral cytotoxic CD4+T-cell function following i.p. injections of Meningococcal Vaccine (Meningo) with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 23A) and low t-cell infiltration tumors (6419c5) (Fig. 23B).

[0037] Figures 24A-24B are graphs showing the percentage of intratumoral granzyme B positive (granzyme B+) of the CD4+T-cell population, indicating intratumoral cytotoxic CD4+T-cell function following i.p. injections of Meningococcal Vaccine (Meningo) with and without anti-CTLA-4 (aCTLA4) in high T-cell infiltrations tumors (2838c) (Fig. 24A) and low t-cell infiltration tumors (6419c5) (Fig. 24B).

[0038] Figures 25A-25D show impaired metastasis of PDAC in a mouse mode. Fig. 25A is a schematic of the experimental design. Briefly, mice with pancreatic cancer were administered via i.p. injection Haemophilus influenzae vaccine (H. flu), Meningococcal conjugate vaccine (MCV) or vehicle control (PBS). Fig. 25B is a graph of the primary tumor weight in grams. Fig. 25C is a graph of the number of tumor foci, illustrating metastasis from the pancreas to the spleen. Fig. 25D is a series of representative images of mouse spleen and pancreas with tumors.

[0039] Figures 26A-26C show impaired tumor growth following administration of H. flu vaccine components tetanus toxid (TT) or polyribosyl ribitol phosphate (PRP). Fig. 26A is a schematic of the experimental design. Briefly, mice with pancreatic cancer were administered via i.p. injection multiple doses of individual H. flu vaccine components, TT, PRP, or vehicle control (PBS). Fig. 26B is a series of representative bioluminescent images showing tumor size on the day of tumor transfer (tDay 0) or 8 days post-tumor transfer (tDay 8). Fig. 26C is a series of images taken on day 13 (tDay 13) of spleen after treatment.

[0040] Figure 27A is a graph of the probably of survival of mice treated with vehicle control (PBS), MCV, or H. flu. Figure 27B is a graph of the number of tumor foci, illustrating the extent of tumor metastasis from the pancreas to the liver.

[0041] Figures 28A-28C show the use of hiFLU vaccine in an orthotopic mouse model of PDAC. Fig. 28A is a graph of the bioluminescence of pancreatic tumor over time, approximating tumor growth in mice. Fig. 28B is a graph of the percentage of cells that are memory T-cells (CD44+ CD62L of the CD8+T-cell population. Fig. 28C is a graph of the percentage of cells that are memory T-cells (CD44+CD62L ) of the CD4+T- cell population.

[0042] Figures 29A-29C show the use of hiFLU in a triple negative breast cancer orthotropic mouse model. Fig. 29A is a graph of the bioluminescence of breast cancer tumor over time, approximating tumor growth in mice. Fig. 29B is a graph of the percentage of cells that are memory T-cells (CD44+CD62L of the CD8+T-cell population. Fig. 29C is a graph of the percentage of cells that are memory T-cells (CD44+CD62L of the CD4+T-cell population.

[0043] Figures 30A-30B are graphs of apoptosis (Fig. 30A) and necrosis (Fig. 30B) after treatment with MCV or H. flu.

[0044] Figure 31 A is a graph of the CD8+ cells to Treg ratio after treatment with MCV and anti-CTLA-4 immune checkpoint inhibitor.

[0045] Figure 31 B is a graph of the IL-10 concentration (picograms per mL (pg / mL)) produced after treatment with MCV, MCV + anti-PD-1 , or MCV + anti-PD-1 + anti-CTLA- 4 (MCV Triple Combo).

[0046] Figures 32A-32B are graphs of the IL-4 concentration (pg / mL) (Fig. 32A) and IL-10 (pg / mL) concentration (Fig. 32B) produced after MCV treatment.

[0047] Figure 33 is a graph of the IL-1 a produced after MCV treatment.

[0048] Figure 34A is a graph of the percentage of M-MDSC of the CD11 b+cell population after MCV treatment.

[0049] Figure 34B is a graph of CCL4 concentration (pg / mL) after treatment with MCV, MCV + anti-PD-1 , or MCV + anti-PD-1 + anti-CTLA-4 (MCV Triple Combo).

[0050] Figure 35 is a graph of vascular endothelial growth factor A (VEGFA) after treatment with MCV or H. flu.

[0051] Figures 36A-36B are graphs of the percentage of high T-cell infiltration tumor cells (Fig. 36A) or low T-cell infiltration tumor cells (Fig. 36B) that survived treatment with Meningo, H. flu, or Pneumo vaccines.Detailed Description

[0052] The embodiments disclosed below are not intended to be exhaustive or to limit the scope of the disclosure to the precise form in the following description. Rather, the embodiments are chosen and described as examples so that others skilled in the art may utilize its teachings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the present document, including definitions, will control.

[0053] The uses of the terms “a” and “an” and “the” and similar references (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ail examples, or exemplary language (e.g., “such as”, “for example”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the embodiments.

[0054] As used herein, the term "amount" refers to "an amount effective" or "therapeutically effective amount" of a composition, e.g., vaccine, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. A "therapeutically effective amount" of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit adesired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient or subject). When a therapeutic amount is indicated, the precise amount of the compositions of the present disclosure to be administered may be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of disease or metastasis, and condition of the subject.

[0055] “Treatment" of a subject refers to any type of intervention or process performed on, or the administration of a microbe-derived vaccine or component thereof to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down, the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a cancer. In some embodiments, “treatment” includes resolution of a particular cancer, including a reduction in one or more symptoms of the cancer and / or a reduction in in the severity of one or more symptoms associated with the cancer.

[0056] Disclosed herein are methods of treating cancer using microbe-derived vaccines. For example, a method of treating cancer may include administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof. The therapeutically effective dose may be administered systemically (e.g., intraperitoneally or intravenously). In some cases, the therapeutically effective dose is administered intraperitoneally or intravenously. In some cases, the therapeutically effective dose is administered systemically to the subject in need thereof using any appropriate administration method, such as intraperitoneally or intravenously.

[0057] Also disclosed herein are methods of increasing the amount of resident memory T-cells (Trms) in a tumor of a subject identified as having a cancer.

[0058] Microbe-Derived Vaccines

[0059] Microbe-derived vaccines as described herein refer to vaccines in which the antigen in the vaccine is derived from a bacteria or virus and stimulates an immune response to produce antibodies against the antigen. Microbe-derived vaccines suitablefor use herein may be any microbe-derived vaccine, i.e. , any vaccine directed toward a microbe, such as a bacteria or a virus. In some cases, a microbe-derived vaccine is a bacterium-derived vaccine. In some cases, a microbe derived vaccine is a virus-derived vaccine. Exemplary microbe-derived vaccines include, but are not limited to, a meningococcal vaccine (e.g., a meningococcal conjugate vaccine (MCV)), a Haemophilus influenzae vaccine, a pneumococcal vaccine, or an influenza vaccine. Suitable vaccines may be FDA-approved or are any other vaccine directed toward one or more antigens of the microbe. In some cases, the microbe-derived vaccine is selected from a meningococcal vaccine (e.g., a meningococcal conjugated vaccine), a Haemophilus influenzae vaccine, and a pneumococcal vaccine.

[0060] Suitable microbe-derived vaccines may include vaccines directed toward a single strain of the microbe (i.e., single valent vaccine), or vaccines directed toward multiple strains of the microbe (i.e., multi-valent).

[0061] Exemplary vaccines include a Haemophilus influenzae vaccine such as Hiberix®, a meningococcal conjugate vaccine such as Bexsero®, or a pneumococcal vaccine such as Prevnar 13®.

[0062] In some cases two or more microbe-derived vaccines (e.g., two, three, four, or more microbe-derived vaccines) may be used in any of the methods described herein. In some cases, two microbe-derived vaccines may be used in any of the methods described herein. In some cases, a meningococcal vaccine (e.g., a meningococcal conjugated vaccine) and a Haemophilus influenzae vaccine are used in any of the methods described herein.

[0063] Formulations of microbe-derived vaccines may be prepared by mixing one or more microbe-derived vaccine with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0064] The microbe-derived vaccines described herein may be unadjuvanted or adjuvanted. For example, an unadjuvanted vaccine may include a microbe-derived vaccine to which an additional adjuvant is not added to the vaccine prior to administration, but which includes the natural adjuvants that are part of the preparation of the vaccine itself. For example microbe-derived cell proteins are considered to be part of an unadjuvanted vaccine.

[0065] Cancers and Subjects Having a Cancer

[0066] Cancers that may be treated using any of the methods disclosed herein include adenocarcinomas. Adenocarcinomas develop in glands that line the inside of organs. Exemplary adenocarcinomas include pancreatic cancer, breast cancer, lung cancer, prostate cancer, and esophageal cancer. In some bases, any of the methods described herein may treat pancreatic cancer or breast cancer.

[0067] Pancreatic cancer can include pancreatic ductal adenocarcinomas (PDAC). Pancreatic cancer often presents with belly pain, loss of appetite, weight loss, jaundice, new-onset diabetes, fatigue, and pain or swelling in an arm or leg. The diagnosis of pancreatic cancer may include imaging tests (such as ultrasound, CT scans, MRI scans, and PET scans), endoscopic ultrasound, biopsies of tumors, blood tests for serum markers (e.g., CA19-9) or genetic markers, and cancer staging (ranging from 0 to 4). These methods help determine the extent of the cancer and guide treatment decisions. PDAC, a highly lethal cancer, has the lowest 5- year survival rate for several reasons (1), including its tendency for the late diagnosis (2), a lack of serologic markers for screening, aggressive local invasion, its early metastatic dissemination, and its resistance to chemotherapy / radiotherapy (3). Just 30% of PDAC patients react to a potent chemotherapy (i.e., FOLFIRINOX (Fluorouracil + Leucovorin + Irinotecan + Oxaliplatin). Despite rapid advances in immunotherapy for most solid cancers, progress in immunotherapy against PDAC has been difficult. This is true for several approaches, most notably immune checkpoint inhibitors (IC Is) and GM-CSF cell-based vaccines(GVAX). Though many immunotherapies have been explored in clinical trials, few have shown significant therapeutic efficacy.

[0068] PDAC is characterized by a low tumor mutation burden and a uniquely immunosuppressive tumor microenvironment (TME), which likely contribute to the lack of significant clinical activity of the immune checkpoint inhibitors ( ICI) targeting CTLA-4 and / or PD-1. Critical obstacles to immunotherapy in PDAC tumors include a high number of tumor-associated immunosuppressive cells and a uniquely desmoplastic stroma that functions as a barrier to T cell infiltration PDAC is a stroma-rich cancers, which supports tumor growth and metastasis and simultaneously serves as a physical barrier to drug delivery.

[0069] Breast cancer may include triple negative breast cancer (TNBC). TNBCs lack or show low levels three common receptors: estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Common symptoms of breast cancer include a breast lump, thickened skin, nipple changes (such as flattening or inversion), skin color changes, and discharge other than breast milk. The diagnostic measures for breast cancer include clinical breast exams, mammograms, breast ultrasounds, breast MRIs, and biopsies. These tests help detect abnormalities, assess tissue samples, and determine the presence of cancer.

[0070] In any of the methods described herein, a subject may be identified as having a cancer using any diagnostic method. In some cases, a subject is identified as having a cancer. In some cases, a subject was previously identified as having a cancer. In some cases, a subject may be identified or was previously identified as having a metastatic cancer or a recurring cancer. In some cases, a subject has not been identified as having a cancer.

[0071] In any of the methods described herein, a subject may have previously been exposed to any of antigens present in the microbe-derived vaccines described herein. For example, a subject may have received a vaccination (e.g., a microbe-derived vaccine as described herein) earlier in life, e.g., as a child. In other cases, a subject may had an infection caused by Neisseria meningitides (also called meningococcus), Haemophilus influenzae, Streptococcus pneumonia, or an Orthomyxoviridae virus (e.g., Influenza virus).

[0072] Administration Routes

[0073] A microbe-derived vaccine may be administered to a subject in need of the treatment via any suitable route for systemic administration. Examples of systemic administration include intravenous and intraperitoneal.

[0074] The term “intravenous administration” refers to the delivery of a microbe- derived vaccine, or pharmaceutical composition including a microbe-derived vaccine, into a subject’s vein using a needle or tube for systemic administration to a subject in need thereof.

[0075] The term “intraperitoneal administration” refers to the delivery of a microbe- derived vaccine, or pharmaceutical composition including a microbe-derived vaccine, directly into the peritoneal cavity.

[0076] In some cases, a microbe-derived vaccine may be administered to a subject in need of the treatment via a route other than intratumoral administration. The term “intratumoral administration” refers to the delivery of a composition into or adjacent to a tumor or cancer and / or immediate vicinity of a tumor or cancer. Multiple injections into separate regions of the tumor or cancer are also included. Furthermore, intratumoral administration includes delivery of a composition into one or more metastases. Methods for intratumoral delivery of drugs are known in the art (Brincker, 1993. Grit. Rev. Oncol. Hematol. 15(2):91 -8; Celikoglu et al., 2008. Cancer Therapy 6, 545-552).

[0077] The administration of a microbe-derived vaccine may be used to accomplish one or more of the following: (a) reducing or limiting tumor size; (b) reducing or limiting tumor growth; (c) reducing or limiting progression, development, and / or metastasis of a cancer; (d) eliminating a tumor; (e) inhibiting, preventing, or reducing the recurrence of a tumor for at least 3 months, at least 6 months, or at least 12 months after administration of the microbe-derived vaccine; (f) promoting an immune response against a tumor; and (g) increasing the amount of resident memory T-cells (Trms) in a tumor of a subject. The effect may be found in the tumor to which the microbe-derived vaccine was administered and / or one or more metastases or other tumors. In some cases, administration of a microbe-derived vaccine may reduce the progression of a cancer. Insome cases, administration of a microbe-derived vaccine may increase the amount of Trms in a tumor of a subject.

[0078] Combination Therapies

[0079] Any of the methods of treatment described herein may also include at least one additional anticancer treatment (e.g., one, two, three, four or more additional anticancer treatments). Exemplary additional anticancer treatments include, but are not limited to, surgical therapy, chemotherapy, radiation therapy, hormonal therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti- angiogenic therapy, cytokine therapy, cryotherapy, radioablation, and a biological therapy. In some cases, the at least one additional cancer treatment is an immune therapy.

[0080] Immune therapies may include monoclonal antibodies directed toward cancer cells, immune checkpoint inhibitors (also called checkpoint blockade immunotherapy), and cancer vaccines. In some cases, the at least one additional anticancer treatment an immune checkpoint inhibitor. In some cases, the at least one additional anticancer treatment is an immune checkpoint inhibitor and / or surgical therapy. In some cases, the at least one additional anticancer treatment is f an immune checkpoint inhibitor, surgical therapy, and / or chemotherapy. In other cases, the at least one additional anticancer treatment is an immune checkpoint inhibitor, surgical therapy, chemotherapy, and / or radiation therapy.

[0081] Immune checkpoint inhibitor therapy (also called checkpoint blockade immunotherapy) targets immune checkpoints, which are key regulators of the immune system. These inhibitors may block checkpoint proteins from binding their partner proteins. When the checkpoint and partner proteins bind together, they send an “off” signal to the T-cells. This can prevent the immune system from destroying the cancer. Immune checkpoint inhibitors work by blocking checkpoint proteins from binding with their partner proteins. This prevents the “off” signal from being sent, allowing the T cells to kill cancer cells. In some embodiments, the microbe-derived vaccine is used in combination with an immune checkpoint inhibitor. As used herein, the term “checkpoint inhibitor” refers to any molecules, including antibodies, fragments thereof, and smallmolecules, that block the immunosuppression pathway induced by one or more checkpoint molecules.

[0082] An immune checkpoint inhibitor may target any one or more of the following checkpoint molecules: cytotoxic T-lymphocyte antigen 4 (CTLA-4) (also known as CD152) (CTLA-4), programmed T-cell death 1 (PD-1 ), programmed T-cell death ligand 1 (PD-L1) (also known as CD279), PD-L2, lymphocyte-activation gene 3 (LAG-3) (also known as CD223), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), CD28, CD122, or 4-1 BB (also known as CD137).

[0083] CTLA-4 is a protein receptor that downregulates the immune system. Nonlimiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, and MDX-101 ) and tremelimumab (formerly ticilimumab, CP- 675,206).

[0084] PD-1 is a trans-membrane protein found on the surface of T cells, which, when bound to PD-L1 or PD-L2 on tumor cells or immune cells, results in suppression of T cell activity and reduction of T cell-mediated cytotoxicity. PD-1 and PD-L1 are immune down-regulators or immune checkpoint “off switches”. Examples of PD-1 inhibitors include, but are not limited to, nivolumab, (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001 , MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C. Examples of PD-L1 inhibitors include Atezolizumab (Tecentriq), Durvalumab (MEDI4736), Avelumab (MSB0010718C), MPDL3280A, BMS935559 (MDX-1105) and AMP-224.

[0085] LAG-3 is an immune checkpoint receptor on the cell surface works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. LAG-3 inhibitors include, but are not limited to, LAG525 and BMS-986016.

[0086] CD28 is constitutively expressed on almost all human CD4+ T cells and on around half of all CD8 T cells. It prompts T cell expansion. A non-limiting example of a CD28 inhibitor is TGN1412.

[0087] CD122 increases the proliferation of CD8+ effector T cells. A non-limiting example of a CD122 inhibitor is NKTR-214.

[0088] CD137 is involved in T-cell proliferation. CD137-mediated signaling is also known to protect T cells, and in particular, CD8+ T cells from activation-induced celldeath. Non-limiting examples of CD137 inhibitors include PF-05082566, Urelumab (BMS-663513) and lipocalin.

[0089] In some cases, a subject was previously administered at least one additional anticancer treatment therapy before receiving a microbe-derived vaccine or pharmaceutical composition thereof.

[0090] Dosing / Administration Regimen

[0091] Selecting a dosing regimen for any of the microbe-derived vaccines described herein or pharmaceutical formulations thereof depends on several factors, including the serum or tissue turnover rate of the subject, the level of symptoms of the subject, the immunogenicity of the subject, and the accessibility of the target cells in the tumor or subject. In certain embodiments, an administration regimen maximizes the amount of therapeutic (e.g., microbe-derived vaccine) delivered to the subject consistent with an acceptable level of side effects. Accordingly, the amount of the microbe-derived vaccine delivered depends in part on the particular subject, the particular tumor, and the severity of the cancer being treated. Guidance in selecting appropriate doses of vaccines, antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991 ) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al., (2003) New Engl. J. Med. 348:601 -608; Milgrom et al., (1999) New Engl. J. Med. 341 : 1966-1973; Slamon et al, (2001 ) New Engl. J. Med. 344:783-792; Beniaminovitz et al., (2000) New Engl. J. Med. 342:613-619; Ghosh et al., (2003) New Engl. J. Med. 348:24-32; Lipsky et al., (2000) New Engl. J. Med. 343: 1594-1602).

[0092] Determination of the appropriate dose is made by a clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount that is less than the optimum dose and the dose is increased by small increments until the desired effect is achieved relative to any negative side effects. Important diagnostic measures includethose of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced, tumor size, and cancer progression.

[0093] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.

[0094] Actual dosage levels of microbe-derived vaccine or pharmaceutical composition including a microbe-derived vaccine, as described herein, may be varied to obtain an amount of the microbe-derived vaccine which is effective to achieve the desired therapeutic response for a particular subject, a particular composition, and a particular mode of administration, without being toxic to the subject or without causing significant side effects. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular composition being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors known in the medical arts (see, e.g., Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001 ) Good Laboratory and Good Clinical Practice, Urch PubL, London, UK).

[0095] One or more therapeutically effective dose of a microbe-derived vaccines or of a pharmaceutical composition including a microbe-derived vaccine can be providedby continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1 -7 times per week using any appropriate route of administration. In some embodiments, a single dose or multiple doses of the microbe-derived vaccine can be administered systemically (e.g., intraperitoneally or intravenously). In some embodiments, dosing of the microbe- derived vaccine or of a pharmaceutical composition including a microbe-derived vaccine can be weekly, every other week, or every two to three weeks. Doses of additional therapeutics used in an additional anticancer treatment can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation.

[0096] In some cases, more than one therapeutically effective dose of a microbe- derived vaccines or of a pharmaceutical composition including a microbe-derived vaccine (e.g., two doses, three doses, four doses, five doses, six, doses, seven doses, eight doses, nine doses, ten doses or more) can be administered to a subject in need thereof. In some cases, a subject has received a therapeutically effect dose of a microbe-derived vaccine or of a pharmaceutical composition including a microbe- derived vaccine. In some cases, a subject has not received a therapeutically effect dose of a microbe-derived vaccine or of a pharmaceutical composition including a microbe- derived vaccine. In some cases, a subject has received a therapeutically effect dose of a microbe-derived vaccine or of a pharmaceutical composition including a microbe- derived vaccine before the subject was identified as having a cancer. In some cases, a subject has not received a therapeutically effect dose of a microbe-derived vaccine or of a pharmaceutical composition including a microbe-derived vaccine before the subject was identified as having a cancer.

[0097] A specific dose protocol or a dosage unit is one involving a dose or dose frequency that avoids significant undesirable side effects. A dosage unit may be at least 0.05 p / kg body weight, at least 0.2 pg / kg, at least 0.5 pg / kg, at least 1 pg / kg, at least 10 pg / kg, at least 100 pg / kg, at least 0.2 mg / kg, at least 1 .0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 40 mg / kg or at least 50 mg / kg (see, e.g., Yang et a!., (2003) New Engl. J. Med. 349:427-434; Herold et al., (2002) New Engl. J. Med. 346: 1692-1698; Liu et al., (1999) J. Neurol. Neurosurg. Psych. 67:451 -456; Portielji et al., (2003) Cancer Immunol. Immunother. 52: 133-144).

[0098] Methods for co-administration or treatment with a second therapeutic agent, e.g., an antibody or chemotherapeutic agent, by way of non-limiting example, are known in the art (see, e.g., Hardman et al., (eds.) (2001 ) Goodman and Gilman's The Pharmacological Basis of Therapeutics, IO.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001 ) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001 ) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%>; at least 40%>, or at least 50%.

[0099] The route of administration for additional therapeutic agents may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraarticular, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et aL, (1983) Biopolymers 22:547-556; Langer et al., (1981 ) J. Biomed. Mater. Res. 15: 167- 277; Langer (1982) Chem. Tech. 12:98-105; Epstein et al., (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang et al., (1980) Proc. Natl. Acad. Sci. USA 77:4030-4034;U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration may also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference their entirety.

[0100] Additional therapies (e.g., microbe-derived vaccine or additional anticancer treatment), which may be administered in combination with any of the microbe-derived vaccines described herein may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hoursto about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 1 1 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the microbe-derived vaccine. The two or more therapies may be administered within one same doctor’s visit.

[0101] The microbe-derived vaccine and the other additional anticancer treatments may be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first microbe-derived vaccine or additional anticancer treatment) for a period of time, followed by the administration of a second therapy (e.g., a second microbe-derived vaccine or additional anticancer treatment) for a period of time, optionally, followed by the administration of a third therapy (e.g., microbe-derived vaccine or additional anticancer treatment) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and / or to improve the efficacy of the therapies.

[0102] The therapies (e.g., microbe-derived vaccine or additional anticancer treatment) of the combination therapies may be administered to a subject concurrently. The term "concurrently" is not limited to the administration of therapies (e.g., microbe- derived vaccine or additional anticancer treatment) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising a microbe-derived vaccine are administered to a subject in a sequence and within a time interval such that the microbe-derived vaccine may act together with the additional therapy(ies) to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy may be administered to a subject separately, in any appropriate form and by any suitable route.

[0103] The microbe-derived vaccine or additional anticancer treatment of the combination therapies may be administered to a subject in the same pharmaceutical composition. Alternatively, the microbe-derived vaccine or additional anticancer treatment of the combination therapies may be administered concurrently to a subject in separate pharmaceutical compositions. The microbe-derived vaccine or additional anticancer treatment may be administered to a subject by the same or different routes of administration.

[0104] In certain embodiments, the microbe-derived vaccine or additional anticancer treatment may be formulated to ensure proper distribution in vivo. For example, treatment of a pancreatic cancer would use a microbe-derived vaccine or an additional anticancer treatment that is formulated to ensure distribution to the pancreas of the subject in need. Similarly, treatment of a breast cancer would use a microbe-derived vaccine or an additional anticancer treatment that is formulated to ensure distribution to the breast(s) of the subject in need. In some embodiments, treatment of a cancer uses a microbe-derived vaccine or an additional anticancer treatment that is formulated to be delivered to an alternative location in the subject in need, e.g., treating a metastatic tumor of a pancreatic cancer in the liver of the subject or treating a metastatic tumor of a breast cancer in the bone of the subject.

[0105] Methods of Increasing the Amount of Resident Memory T-cells (Trms)

[0106] Also disclosed herein is a method of increasing the amount of Trms in a tumor of a subject identified as having a cancer. Without being limited by theory, a subject’s immune system may be primed against antigens, including antigens of cancer cells, and swiftly recalled when growth of a tumor expressing antigens similar in sequence and structure are identified. Developing long-term protective immunity may limit or prevent relapse and infiltration of tumors with T-cells with a resident memory (Trm) phenotype correlates with improved survival rate (see Examples).

[0107] Methods of increasing the amount of Trms in a tumor or in a sample of a tumor of a subject in need thereof (i.e., a subject having cancer, a subject identified as having cancer, a subject previously identified as having cancer, or a subject not identified as having cancer) include administering a therapeutically effect dose of one ormore of any of the microbe-derived vaccines described herein to the subject. The amount of Trms may be compared to any appropriate reference level of a Trms in a tumor of the same cancer. For example, a reference level of Trms may include a reference level of Trms from a matched sample from the same tumor before treatment, from a different tumor of the same subject before the same treatment, or from a different tumor of a different subject having the same cancer before the same treatment. A reference level of Trms may also include a literature value of an average number of Trms in a tumor or tumor sample.

[0108] Methods of increasing the amount of Trms in a tumor (e.g., in a sample of a tumor) of a subject in need thereof may also include identifying the subject as having a cancer, as described herein. Methods of increasing the amount of Trms in a tumor (e.g., in a sample of a tumor) of a subject in need thereof may also include determining the amount of Trms in the tumor after administering the therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor. Methods of increasing the amount of Trms in a tumor (e.g., in a sample of a tumor) of a subject in need thereof may also include determining a reference level of Trms in a tumor (e.g., a sample of a tumor). The amount of Trms in a tumor or tumor sample may be determined using any appropriate method. For example, the amount of Trms in a tumor or tumor sample may be measured using flow cytometry of a sample of tumor cells to identify CD44+ CD62- T-cells. In some cases, the amount of Trms in a tumor or tumor sample may be measured as described in the Examples section of this application.

[0109] In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 5% higher (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) after administration of the microbe-derived vaccine or pharmaceutical composition thereof. In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 5% higher (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) after administration of the microbe- derived vaccine or pharmaceutical composition thereof compared to the amount of Trms in the tumor before administration of the microbe-derived vaccine. In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 5% higher (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) afteradministration of the microbe-derived vaccine or pharmaceutical composition thereof compared to the amount of Trms in a tumor from a different subject that was not administered the microbe-derived vaccine or pharmaceutical composition thereof.

[0110] In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 1 .2-fold higher (e.g., at least 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1 .7-fold, 1 .8- fold, 1 .9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher) after administration of the microbe-derived vaccine. In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 1 .2-fold higher (e.g., at least 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher) after administration of the microbe-derived vaccine compared to the amount of Trms in the tumor before administration of the microbe-derived vaccine. In some cases, the amount of Trms in a tumor (e.g., a sample of a tumor) is at least 1 .2-fold higher (e.g., at least 1 .3-fold, 1 .4-fold, 1 .5-fold, 1 .6-fold, 1 .7-fold, 1 .8-fold, 1 .9-fold, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold or higher) after administration of the microbe-derived vaccine compared to the amount of Trms in a tumor from a different subject that was not administered the microbe-derived vaccine or pharmaceutical composition thereof.

[0111] In some embodiments, a method of increasing the amount of Trms in a tumor (e.g., in a sample of a tumor) of a subject in need thereof includes systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor, wherein the amount of Trms is compared to a reference level of Trms in a tumor of the same cancer, wherein the cancer is a pancreatic ductal adenocarcinoma (PDAC), and wherein the amount of Trms in the tumor is 2-fold higher than a reference level of Trms in a tumor of the same cancer. In some cases, the reference level of Trms is the amount of Trms in a tumor from a different subject that was not administered the microbe-derived vaccine or pharmaceutical composition thereof.Examples

[0112] The following Materials and Methods are used throughout the following examples, unless otherwise indicated.

[0113] Mice: C57BU6 mice aged 6 to 8 weeks, used to generate the 6419c5 and KPC-luc tumor models, were purchased from The Jackson Laboratory. Pdx1 -Cre x LSL- KrasG12Dx LSL-TP53R172H(KPC) mice were obtained. Male mice were used for all experiments conducted with C57BL / 6 mice, and female mice were used for all experiments with KPC mice. Animals were housed in specific-pathogen-free facilities.

[0114] Cell Lines: The 6419c5 cell line was isolated from a late-stage primary PDAC tumor in a KPC mouse (KrasG12D / +;LSL-Trp53R172H / +;Pdx1 -Cre) and characterized as producing low T cell infiltrating tumors. See, for example Li etal., Immunity. 2018 Jul 17;49(1):178-193.e7. The KPC-luc cell line was generated from a tumor in a Pdx1 -Cre x LSL- rasG12Dx LSL-TP53R172Hmouse followed by luciferase gene insertion into the cell line through lentiviral transduction16. Both cell lines were cultured using DMEM supplemented with 2 mM glutamine, 10% FBS, 100 units / mL penicillin, and 100 mg / mL streptomycin. The HEK-Blue TLR reporter cell lines were stably transfected with plasmids containing a single murine TLR gene and a NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) gene to measure TLR stimulation. The expression of TLR and SEAP plasmids was maintained through supplementing the aforementioned complete DMEM medium with 30 pg / mL blasticidin and 100 pg / mL zeocin as selection antibiotics. Measurement of TLR activation was assessed.

[0115] Tumor Development: For experiments using the 6419c5 tumor model, 5 x 105cells suspended in PBS were subcutaneously administered into the right flank of C57BL / 6 mice. Animals were anesthetized with isoflurane for the procedure. Changes in tumor growth were monitored with caliper measurements in two perpendicular experiments. For experiments utilizing the KPC-luc orthotopic model, C57BL / 6 mice were initially anesthetized with a ketamine / xylazine cocktail (90 mg / kg and 10 mg / kg, respectively). Anesthesia was maintained through isoflurane inhalation, and animals were placed on their right side, exposing the area of the spleen in which the hair surrounding the area had been removed the day prior. Povidone iodine solution followed by 70% isopropyl alcohol was used to sterilize the area prior to incision. A 1 cm incision was made through the skin and musculature, exposing the underlying organs. The pancreas and spleen were gently pulled out of the incision site using a sterile cotton tipped applicator. Once situated, 8 x 104KPC-luc cells in a 30% Matrigel solution wereinjected into the tail of the pancreas. After injection, the pancreas and spleen were returned to their correct anatomical position and musculature and skin were sutured to close the incision. Changes in tumor growth was monitored through intraperitoneal injection of 150 mg / kg D-luciferin, followed by bioluminescence measurement using an in vivo imaging system (I VIS), twice weekly.

[0116] Survival Studies: Following completion of the treatment regimen, KPC and KPC-luc tumor bearing mice were monitored and weighed for signs of decline in health, at least twice a week. Monitoring was continued until animals had succumbed to disease, or had met health-related endpoints requiring euthanasia, as per IAUCUC policies.

[0117] In Vivo Depletion Studies: In vivo depletion of CD4+ T cells, CD8+ T cells, and NK cells were conducted using anti-CD4 (clone GK1 .5), anti-CD8 (clone YTS169.4), and anti-NK1.1 (clone PK136) purchased from BioxCell. 300 ug of anti- CD4, anti-CD8, and anti-NK1 .1 was administered to KPC-luc tumor-bearing mice via intraperitoneal injection. The respective isotype for each depletion antibody was administered to control groups.

[0118] Vaccine and Vaccine Components: The FDA-approved Hiberix® vaccine indicated for immunization against Haemophilus influenzae type b was manufactured by GlaxoSmithKline.

[0119] HibVx (also referred to as H. flu) was reconstituted with PBS as described in the FDA insert. Once reconstituted, 50 ul of undiluted vaccine was administered intratumorally. Experiments utilizing intraperitoneal administration 100 ul of vaccine was used. For the component studies, the formaldehyde-inactivated tetanus toxoid (TT) derived from Clostridium tetani was purchased from Sigma Aldrich and Enzo Life Sciences. The Haemophilus b capsular polysaccharide, polyribosyl-ribitol-phosphate (PRP) was purchased from Creative Diagnostics. Both components were reconstituted in PBS to deliver a 1 ug dose of PRP or 2.5 ug TT was administered via intraperitoneal injection, the same amount found in a 100 ul dose of HibVx. PBS was used as a control for all vaccine and component studies.

[0120] H. flu’s components for (50 ul dose):-1 mcg of purified capsular polysaccharide conjugated to approximately 2.5 mcg of tetanus toxoid (Tetanus toxin from Clostridium tetani) -1.26 mg of lactose- <0.05 mcg of residual formaldehyde

[0121] The FDA- approved Meningococcal Group B Vaccine (Bexsero®) vaccine indicated for immunization against four type of Neisseria meningitidis bacteria, manufactured by GlazoSmithKline. MCV was reconstituted with PBS as described in the FDA insert. 50 ul of undiluted vaccine was administered intratumorally. Experiments utilizing intraperitoneal administration 100 ul of vaccine was used.

[0122] MCV Components (50 uls of dose)-5 ug Neisserial adhesion A recombinant protein-5 ug Neisserial heparin binding antigen-5 ug Factor H binding protein-2.5 ug outer membrane vesicles-0.15 mg aluminum hydroxide-0.3125 mg NaCI-0.0776 mg Histidine-1 mg sucrose @ pH 6.4-6.7

[0123] The FDA-approved Pneumococcal 13-valent Conjugate vaccine (Prevnar 13®, also referred to as PV or Pneumo) indicated for immunization against many types of pneumococcal bacteria, manufactured by Wyeth Pharmaceuticals LLC (subsidiary of Pfizer). PV was reconstituted with PBS as described in the FDA insert. 50 ul of, undiluted vaccine was administered intratumorally. Experiments utilizing intraperitoneal administration 100 ul of vaccine was used.

[0124] PV components (50 uls of dose)0.22 of each of Streptococcus pneumoniae serotypes 1 , 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F Saccharides,0.44 ug of 6B saccharides,0.3 ug CRM197 carrier protein,1 ug of polysorbate 80,29.5 ug succinate buffer,12.5 ug aluminum phosphate adjuvant.

[0125] Flow Cytometry: Tumors were prepared into a single cell suspension as previously described. See, for example, Newman et al., Proc Natl Acad Sci U S A. 2020 Jan 14; 117(2) :1119-1 128. To identify viable cells, Live / Dead Fixable Aqua Dead Cell Stain Kit (405 nm excitation) was added to antibody staining cocktails. To identify immune cells in the tumor samples, anti-CD45 antibodies were used. Using the CD45+ population, T cells were identified using a CD3-targeting antibody, CD8+ T cells were identified using CD3 and CD8-specific antibodies, and CD4+ T cells were identified using CD3 and CD4 specific antibodies. Natural killer cell populations were identified using NK1 .1 and CD3 (negative gating) antibodies. Tumor associated macrophage populations were identified using CD1 1 b and F4 / 80 targeting antibodies. Myeloid derived suppressor cells were identified using CD1 1 b and Gr-1 specific antibodies. Extracellular staining cocktails also included antibodies specific to CD62L, CD44, LAG- 3, and CD69. The amount of each antibody used for extracellular staining was determined by the manufacturer’s instruction. For intracellular staining, samples were permeabilized and fixed using Cytofix / Cytoperm per the manufacturer’s instructions prior to staining with FOXP3, Granzyme B, and T-bet targeting antibodies. The respective isotype controls were run in simultaneous with antibody-stained samples. All flow cytometry was completed using the BD LSRFortessa. Flow cytometry analysis was then analyzed using FlowJo (version 10.10).

[0126] Immunohistochemical / lmmunofluorescent Staining: Resected tumor samples were placed in 10% formalin for 24 hours followed by 70% ethanol for at least 24 hours. Samples were then dehydrated with an increasing ethanol gradient and embedded in paraffin blocks. 5 urn sections were cut from the blocks using a microtome and placed on slides. Deparaffinization was completed through two xylene washes and rehydrated with a decreasing ethanol gradient. Overnight incubation in tri-sodium citrate buffer at 60°C was subsequently used for antigen retrieval. The slides were then blocked with 5% bovine serum albumin, and incubated with antibodies targeting CD3, CD4, or CD8. Hematoxylin and eosin staining was completed using an H&E staining kit purchased from Abeam, following the manufacturer’s instructions.

[0127] Statistical Analysis: Statistical analyses were completed with GraphPad Prism Version 10.1 .1 . A two-way ANOVA with Tukey correction was used to compare data with multiple timepoints. For a single comparison between two groups, a paired Student’s t-test was used. For comparison between more than two groups, a 1 -way ANOVA was used. To ascertain statistical significance in survival studies, a Log-rank (Mantel-Cox) curve was generated. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01 , ***P < 0.001 , and ****P < 0.0001 .Example 1. Determining Toll-Like Receptors Are Stimulated In Vitro by Meningococcal Conjugate Vaccine (MCV) in PDAC.

[0128] To test the hypothesis that administration of MCV enhances intratumoral T- cell proliferation and cytokine production to elevate anti-tumor responses, a series of in vitro experiments were performed to assess toll-like receptor (TLR) activity. TLRs are receptors on innate immune cells, including on dendritic cells, that recognize a variety of microbial structure and play a role in T-cell stimulation. The HEK-Blue-mTLR cell line (Invivogen), engineered to express various mouse TLRs, and its parental cell line, Null2-k (Invivogen), which does not express any mouse TLRs, were propagated and used to determine TLR activity, as per the manufacturer’s instructions. In HEK-Blue- mTLR cells, secreted embryonic alkaline phosphatase (SEAP) is produced upon stimulation with a TLR7 ligand and real-time detection of SEAP was conducted utilizing HEK-Blue Detection medium (Invivogen). Briefly, treatment with MCV or treatment with control were plated in triplicate: 20 pl of MCV (MOI = 1 ), positive control TLR agonist, and negative control, PBS (1x). Then, the respective cell lines (5 x 104cells in 180 pl of medium per well) were added to the appropriate wells. The plate was incubated for 24 hours at 37°C and 5% CO2 and analyzed using a Cytation 3 (Biotek) plate reader at OD620 nm. MCV stimulated TLR2, TLR4, TLR7 and TLR9 (Fig. 1 B), indicating that MCV treatment may be able to activate T-cell response in tumors in vivo.Example 2. Slowing PDAC Tumor Growth in Vivo with Meningococcal Conjugate Vaccine (MCV) Treatment.

[0129] To test the hypothesis that the treatment of pancreatic ductal adenocarcinoma (PDAC) with MCV would overcome the excessive fibrosis characteristic of PDAC tumors and exert a protective antitumor response, a murine pancreatic tumor mouse model was used. Briefly, 6-8-week old C57BL / 6 mice were subcutaneously (s.c.) implanted with 1.0 x 106cells of either low T-cell infiltration clone (6419c5) or high T-cell infiltration clone (2838c) to generate PDAC tumors. The resulting PDAC tumors were treated with 50pls of MCV (also called Meningo) or phosphorbuffered saline (PBS) as a control by intraperitoneal (i.p.) injection on days 9, 10, and 11 post-implantation. Tumor area (mm2) was measured approximately every other day starting at day 6 and tumors were harvested after two days of regression for two consecutive days. Mice with tumors derived from low T-cell infiltration or high T-cell infiltration clones treated with MCV had significantly smaller tumors throughout the experiment, showing that MCV treatment inhibited PDAC tumor growth and therefore reduced PDAC progression (Figs. 2A-2C).Example 3. Intraperitoneal Administration of Asplenia Vaccine Reduces PDAC Progression in Mice.

[0130] Asplenia vaccines involves three separate vaccines, which are approved by FDA.• Meningococcal Conjugated Vaccine (Meningo)• Haemophilus influenzae type B Vaccine (H. flu)• Pneumococcal Vaccine (Pneumo)

[0131] The effect of treatment with each of the three separate asplenia vaccines (Meningo, H. flu, and Pneumo) were assessed in a mouse model. Briefly, 6-8-week old C57BL / 6 mice were subcutaneously (s.c.) implanted with 1 .0 x 106cells of either low T- cell infiltration clone (6419c5) or high T-cell infiltration clone (2838cs) to generate pancreatic tumors. Using both low and high T-cell infiltration clones exemplifies possible heterogeneity of tumors of different subjects and provides more robust experimentalresults. Such heterogeneity can be cause by, or partially caused by, inherent differences in a subject’s immune system. Mice were intratumorally administered three doses of each of the vaccines individually, or with control PBS. Intratumoral administration of each of the asplenia vaccines (50 pls injected) administered individually significantly limited the size of tumors in tumors derived from the high T-cell infiltration clone as compared to tumors treated with PBS, and reduced PDAC progression (Fig. 3A). In tumors derived from the low T-cell infiltration clone, intratumoral administration of the Meningo vaccine significantly limited the size of tumors, and reduced PDAC progression, as compared to the control (PBS), while intratumoral administration of the H. flu vaccine and the Pneumo vaccine did not significantly limit the size of tumors and did not reduce PDAC progression (Fig. 3B). However, systemic administration via i.p. injection individually of each of the three asplenia vaccines significantly limited the size of tumors derived from the low T-cell infiltration clone and high T-cell infiltration clone as compared to tumors treated with the control (PBS), and therefore reduced PDAC progression (Fig. 4A-4B).

[0132] After i.p. administration of each of the asplenia vaccines, tumors were isolated and processed to analyze the intratumoral immune cells using flow cytometry. Tumors showed an increased the percentage of CD8+T-cells of the T-cell population (Fig. 5A-5B), the percentage of CD4+T-cells of the T-cell population (Fig. 6A-6B), and the percentage of natural killer (NK) cells of the CD45+ cell population (Fig. 7A-7B), indicating increased intratumoral infiltration of each of these three immune cell populations, which helped generate an anti-tumor tumor microenvironment. In addition, i.p. administration of the Meningo vaccine reduced the percentage of myeloid derived suppressor cells (MDSCs) of the CD11 b+cell population (Fig. 8A-8B). As MDSCs are pro-tumor cells, a reduction in the percentage of MDSCs also helped generate an antitumor tumor microenvironment.

[0133] Also, i.p. administration of the Meningo vaccine increased the percentage of Granzyme B secreting CD8+T-cells in the low T-cell infiltration tumor (Fig. 8C-8D) and increased the percentage of Granzyme B secreting CD4+T-cells in the low T-cell infiltration tumor (Fig. 8E-8F). The percentage of CD11c+MCHII+cells (Macrophages)of the MCHII+ cell population were unchanged (Fig. 8G-8H), as were F4 / 80+CD11b+(Macrophages) of the CD45+cell population (Fig. 8I-8J).

[0134] Also, i.p. administration of the Meningo vaccine decreased the survival of high T-cell infiltration tumor cells (Fig. 36A) and low T-cell infiltration tumor cells (Fig. 36B) compared to vehicle control (PBS).

[0135] Taken together, these data showed that i.p. administration of each of the asplenia vaccines (Meningo, H. flu, and Pneumo) limited pancreatic tumor growth and reduced PDAC progression by generating an anti-tumor tumor microenvironment.Example 4. In vivo analysis of Haemophilus influenzae Vaccine (H. flu) as Immunotherapy for PDAC in a Mouse Model.

[0136] To test the hypothesis that treatment with the Haemophilus influenzae vaccine (H. flu) would induce regression of PDAC tumors, in vivo experiments were performed using a KPC-Luc orthotopic murine model. The mice received a full cycle of treatment with H. flu in which 50 pls of H. flu was administered via i.p. injections.

[0137] Figure 9A depicts the development of orthotropic murine model and images representing Day 0 and Day 10 after treatment (tDays). Figure 9B showed lower tumor bioluminescence in tumors treated with H-Flu compared to tumors treated with PBS, indicating tumor regression from H. flu treatment.

[0138] Using flow cytometry, the orthotropic tumors were examined to obtain a more detailed image of the tumor microenvironment and T-cell population characteristics. The tumor was more immune-active than immune-suppressive, as indicated by the increase in the percentage of CD4+T cells (Fig. 10A) and CD8+T-cell (Fig. 10B). In addition, the ratio between CD8+T-cells and regulatory T-cells (Tregs) increased, consistent with the increase in the percentage of CD8+T-cells (Fig. 10C).

[0139] Taken together, these data showed that immune recognition of H. flu limited tumor growth, reduced PDAC progression, and generated an anti-tumor tumor microenvironment by stimulating the intratumoral immune cells.Example 5. Using Microbe-Derived Vaccines to Activate Anti-Cancer Resident Memory T-cells (Trms) and Treat PDAC in Mouse Models.

[0140] This example illustrates that using different microbe-derived vaccines to finetune the generation of resident memory T-cells (Trm) or circulating memory T-cells are sufficient for anti-tumor immunity, and that Trm cells improves anti-tumor efficacy.

[0141] C57BL / 6 mice were anesthetized with a ketamine / xylazine cocktail (90 mg / kg and 10 mg / kg, respectively). Anesthesia was maintained through isoflurane inhalation, and animals were placed on their right side, exposing the area of the spleen in which the hair surrounding the area had been removed the day prior. Povidone iodine solution followed by 70% isopropyl alcohol was used to sterilize the area prior to incision. A 1 cm incision was made through the skin and musculature, exposing the underlying organs. The pancreas and spleen were gently pulled out of the incision site using a sterile cotton tipped applicator. Once situated, 8 x 104KPC-luc cells in a 30% Matrigel solution were injected into the tail of the pancreas. After injection, the pancreas and spleen were returned to their correct anatomical position and musculature and skin were sutured to close the incision. Changes in tumor growth was monitored through intraperitoneal injection of 150 mg / kg D-luciferin, followed by bioluminescence measurement using an in vivo imaging system ( I VIS), twice weekly. In a KPC-Luc orthotropic mice model for PDAC, the effect of Meningococcal Conjugative Vaccines (MCV) and Haemophilus influenzae (H. flu) on intratumoral memory T-cells were tested. Mice were intraperitoneally administered MCV or H. flu vaccine, or PBS control, on days 6, 7, and 8, with 4-5 mice per treatment group (Fig. 11 B). Tumor size was measured with bioluminescence each day and quantified. Survival was also measured. Treatment with either MCV or H. flu limited tumor growth rates (Figs. 11 C-11 D) and increased survival (Fig. 11 E).

[0142] In addition, single-cell suspensions were generated from harvested tumors and analyzed by flow cytometry for infiltration to analyze the infiltrating immune cell populations. Single cell suspensions from tumors were stained with anti-mouse lineagespecific antibodies of phenotypic markers of memory T-cells (Table 1 ). Trm are characterized by stable surface expression of CD69 and an enhanced effector abilitythat provides a tissue-wide alert state against local reinfection, and are a subset of memory CD8+and CD4+T cells that can provide long-term immune protection in peripheral tissues. A decrease of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs) was observed in the KPC- Luc orthotopic mutant mice model of PDAC (Fig. 11 F-11 H), while an increase in CD4+ T-cells, CD8+ T-cells, NK, cells, and CD69+CD8+T-cells (Trms) was observed was observed in the KPC-Luc orthotopic mutant mice model of PDAC (Figs. 111-11 L).

[0143] Taken together, these data showed that MCV and H. flu treatment limited tumor growth, prevented PDAC progression, and induced alterations in the tumor microenvironment, fostering anti-tumor immune responses by enhancing the influx of anti-tumor immune cells while reducing the immunosuppressive (pro-tumor) immune cells through targeted inhibition of MDSCs, Tregs, and TAMs.Table 1 . Exemplary Phenotypic markers of memory T cells in tumor tissueExample 6. Pre-treatment with Microbe-Derived Vaccines Limits Tumor Growth.

[0144] Using the same C57BL / 6 mice orthotopic mouse model of PDAC as in Example 5, the effect of pre-treatment to microbe-derived vaccine was tested. Briefly C57BL / 6 mice were administered MCV or H. flu vaccine twice, 8 days and 1 day before challenge with the KPC-luc orthotopic PDAC tumor. Following tumor challenge, tumorbearing mice were again administered MCV or H. flu vaccines on days 6, 9, 11 , 13, 15, and 17 post-tumor challenge (Fig. 12A). Bioluminescence imaging to track tumor size was performed throughout the experiment and mice were dissected and tumors were processed for flow cytometry on day 20.

[0145] Pre-treatment with either MCV or H. flu vaccine limited tumor growth (Fig. 12B-12C). Single-cell suspensions were generated from harvested tumors and analyzed by flow cytometry to assess tumor infiltration. Results are shown in Figs. 12D- 121. Administration of microbe-derived derived vaccines developed tissue-resident memory T-cells (Trms), which are a subset of memory CD8+and CD4+T cells that can provide long-term immune protection in peripheral tissues. Trms have both effector (Fig 12D and 12G) and memory T-cell functions (Fig. 12E, 12H, 12J, and 12K), where effector T-cells actively combat infections or diseases, and memory T-cells facilitate faster responses to previous threats, such as infections or diseases.

[0146] In another experiment using the same experimental protocol (Fig. 25A), administration of MCV significantly limited primary tumor growth, while administration of H. flu vaccine only modestly limited primary tumor growth, as measured by primary tumor weight (Fig. 25B). Furthermore, administration of H. flu or MCV resulted in fewer tumor foci on the pancreas and spleen (Fig. 25C-25D).Example 7. Combination of Microbe-Derived Vaccines Alters Intratumoral Immune Cell Populations.

[0147] Briefly, 6-8-week old C57BL / 6 mice were subcutaneously (s.c.) implanted with 1 .0 x 106cells of either T-cell low clone (6419c5) or T-cell high clone (2838cs) to generate PDAC tumors. After treatment with vehicle control (PBS), Haemophilus influenzae vaccine (H. flu), Meningococcal vaccine (Meningo), or the combination of H.flu and Meningo, tumors were isolated and analyzed to characterize intratumoral immune cell populations. Mice treated with a combination of H. flu and Meningo showed a number of immune cell population changes. A schematic is shows in Fig. 12J.

[0148] Following treatment with MCV, H. flu or a combinatorial treatment of MCV + H. flu, tumor size decreased in High T-cell infiltration tumors (Fig. 12K) and in Low T-cell infiltration tumors (Fig. 12L). Also, intratumoral Treg populations were decreased (Figs. 13A-13B). As Tregs suppress the pro-tumor tumor microenvironment and treatment with the vaccine combination reduced the Tregs populations size, the tumor microenvironment because more anti-tumor. Second, treatment with the vaccine combination increased the percentage of CD4+T-cells and CD8+T-cells releasing granzyme B, a tumor-killing agent, and therefore enhanced the cytotoxic T-cell function (Figs. 14A-14B and 15A-15B). Third, treatment with the vaccine combination increased the ratio of CD8+T-cells to Treg cells and a higher ratio is indicative of an anti-tumor microenvironment (Figs. 16A-16B). Fourth, treatment with the vaccine combination increased CTLA-4 expression on CD8+T-cells, an immune checkpoint marker (Figs. 17A-17B), but did not increase PD-1 expression CD8+T-cells.Example 8. Slowing PDAC Tumor Growth in Vivo with Meningococcal Conjugate Vaccine (MCV) and CTLA-4 Immune Checkpoint Inhibition (aCTLA-4) Combination Treatment.

[0149] To test the hypothesis that the treatment of PDAC with MCV in combination with immune checkpoint therapy would overcome the excessive fibrosis characteristic of PDAC tumors and exert a protective antitumor response, a murine pancreatic tumor mouse model was used. Briefly, 6-8-week old C57BL / 6 mice were subcutaneously (s.c.) implanted with 1.0 x 106cells of either T-cell low clone (6419c5) or T-cell high clone (2838cs) to generate PDAC tumors. Four treatment groups consisting of 10 mice per group were used: Group 1 : no treatment control; Group 2: anti-CTL4 + PBS (vehicle); Group 3: hamster IgG isotype control + MCV (depletion antibody control); and Group 4: anti-CTLA4 + MCV. When the tumors reach 2mm2, mice receive 50pls of MCV or vehicle control. After three treatments with MCV, mice were treated with 250pg / kganti-CTLA4 or IgG isotype control intraperitoneally (i.p.) twice (Fig. 19A). Tumor area (mm2) was measured approximately every other day starting at day 10 and tumors were harvested after two days of regression for two consecutive days. The harvested subcutaneous tumors treated to disassociate cells to facilitate intratumoral immune cell analysis by flow cytometry. Mice with either T-cell low or T-cell high clone PDAC tumors that were treated with MCV had significantly smaller tumors throughout the experiment, illustrating that MCV + anti-CTLA4 combinatorial treatment slowed PDAC tumor growth (Figs. 19B-19C), that MCV+ anti-PD-1 combinatorial treatment slowed PDAC tumor growth (Figs. 19D-19E), and that MCV + anti-CTLA-4 + anti-PD-1 combinatorial treatment slowed PDAC tumor growth (Figs. 19F-19G).

[0150] In addition, the intratumoral immune cell populations were characterized. The combination of MCV and aCTLA-4 treatment increased CD4+T-cell influx in tumors (Figs. 20A-20B), increased CD8+T-cell influx in tumors (Figs. 21A-21 B), and increased infiltration of natural killer (NK) cells in tumors (Figs. 22A-22B). The combination of MCV and aCTLA-4 treatment also enhanced effector T-cell function by increasing the percentage of granzyme B+cells of the CD4+ T-cell population (Figs. 23A-23B), and of the CD8+ T-cell population (Figs. 24A-24B). .

[0151] Treatment with MCV and MCV+ anti-CTLA-4 increased the ratio of CD8+ T- cells to Tregs (Fig. 31 A). MCV as a monotherapy and in combination with ICI decreased IL-10 production (Fig. 31 B, 32B). In many tumor models, IL-10 is an immunosuppressive, pro-tumor cytokine. The reduction in IL-10 can potentially be explained by MCV therapy decreasing the amount of Tregs as Tregs produce large amounts of IL-10 (with respect to CD8 T cells). It is likely MCV is increasing CD8 T cells in the tumor while decreasing Tregs (and less intratumoral IL-10).

[0152] MCV treatment also decreased IL-4 production (Fig. 32A). The decrease in IL-4 via MCV may have decreased the pro-survival, anti-apoptotic effect of IL-4 on cancer cells, (i.e., it interacts directly with the PDAC tumor cells) and / or the decrease in IL-4 via MCV prevented CD4+T helper cells from skewing the naive cells into a Th2 phenotype. Th2 cells can have a more immunosuppressive, pro-tumorigenic effect in cancer, in part by production of immunosuppressive cytokines including IL-10. In eithercase, a decrease in IL-4 production would indicate a more anti-tumor tumor microenvironment.

[0153] MOV treatment also decreased IL-1 a secretion (Fig. 33). IL-1 a is a cytokine that contributes to extra cellular matrix (ECM) production and is secreted from fibroblasts, including cancer-associated fibroblasts. Because MCV decreases IL-1 a MCV may decrease cancer cell stroma, allowing anti-tumor immune cells to more easily access the tumor.

[0154] MCV treatment also decreased intratumoral CCL4 and MDSCs (Figs. 34A- 34B). MCV treatment in combination with ICI (either anti-PD-1 or anti-PD1 + anti-CTLA- 4) also decreased intratumoral CCL4. CCL4 is a marker associated with cancer cell proliferation, and attracts MDSCs and pro-tumor macrophages.

[0155] H. flu decreased VEGFA production, and likely indicated a decrease in tumor- associated vascularity. VEGF is a pro-tumor growth factor associated with angiogenesis.Example 9. H. flu Vaccine Components Impaired Pancreatic Tumor Growth in a Mouse Model.

[0156] TT is an inert tetanus toxin that has no toxicity or adverse effects. Because of its potent anti-inflammatory, immunomodulatory, and anti-angiogenic qualities — which include the development of anti-tumor natural killer T cell subsets — it was considered for repurposing as an anti-cancer medication. Polyribosyl ribitol phosphate (PRP) has a key role in the virulence of the Haemophilus influenzae, and it is the main source of serum bactericidal activity and rising antibody levels in the H. flu. See, for example, Zepp, et al. European Journal of Pediatrics 156, 18-24 (1996). For children under the age of two, it is recognized to be safe and can serve as adjuvant for TT to develop anti-tumor activity.

[0157] To test the anti-cancer properties of TT and PRP, an orthotopic mouse model was used (C57BL / 6 mouse with KPC-luc orthotopic PDAC challenge). Briefly, mice were administered via i.p. injection either TT, PRP, or vehicle control (PBS) for five consecutive days, starting on day 6 following tumor challenge (Fig. 26A).Bioluminescence imaging was performed on day 0 and day 8 to assess tumor growth. Following treatment and conclusion of the experiment, spleens were removed to visuallyassess the size of tumors. Both TT and PRP treatment inhibited tumor growth according to bioluminescence imaging (Fig. 26B) and visual inspection of tumor size (Fig. 26C).Example 10. Administration of Microbe-Derived Vaccines Decreased Tumor Metastasis.

[0158] Figs. 27A-27B show the results of administering via i.p. injection vehicle control (PBS), MCV, or H. flu in a KPC mutant mouse model (a genetic model of PDAC). Survival was increased (Fig. 27A) and metastasis of tumor to the liver was decreased (Fig. 27B). As tumors begin to develop in this mouse model after 60 days, treatment began on day 120. Mice were intraperiontineally administered 50 pl doses of the indicated vaccine for 5 consecutive, beginning on day 120.Example 11. Administration of hiFLU in an orthotopic model of PDAC.

[0159] Figs. 28A-28C show the results of administering via i.p. injection hiFLU in an orthotopic model of PDAC. Tumor progression was slowed (Fig. 28A) and the percentage of intratumor memory T-cells of the CD8+T-cell population (Fig. 28A) and of the CD4+T-cell population increased.Example 12. Administration of hiFLU in an orthotopic model of Triple Negative Breast Cancer (TNBC).

[0160] Figs. 29A-29C show the results of intratumorally administering 50 pl of hiFLU in an orthotopic model of TNBC. Tumor progression was slowed (Fig. 28A) and the percentage of intratumor memory T-cells of the CD8+T-cell population (Fig. 28A) and of the CD4+T-cell population increased.Example 13. Determining Altered T-Cell Receptors (TCR) and T-cell Subpopulations Induced in Vivo by Meningococcal Conjugate Vaccine (MCV) in PDAC.

[0161] To test the hypothesize that treatment of PDAC with MCV alters the TCR repertoire via TLR signaling, a series of in vivo tests can be used to identify altered TCR repertoires and the Ikzf2+ T-cell subpopulation (can generate novel T-cells) induced following MCV treatment. Briefly, in a mouse model of PDAC, control (PBS-injected) tumor and tumors treated with MCV are analyzed to determine the mean representation of tumor-associated TCR clones. Productive TCR clones are derived from MCV treated and control tumors and compared. TCR clones from control tumors are considered “tumor associated.” Single-cell RNA sequencing (scRNA-seq) is performed using 10X Genomics Single Cell 5’ Platform. Samples of treated and control tumors will be enzymatically dissociated (Miltenyi), filtered through a 70-micron cell strainer, pelleted after centrifugation at 300g and resuspended in DAPI-FACS buffer (PBS, 0.04% BSA). Samples are sorted, and viable singlets are gated based on scatter properties and DAPI exclusion. Approximately 3000 cells are pelleted and resuspended in PBS, and cells undergo single cell droplet-based capture on 10X Chromium instruments according to the 10X Genomics Single Cell 5' Platform protocol in order to sequence TCRs. Tumor-associated TCR clones represented in at least one control tumor (n) and detected in a greater number of MCV-treated tumors (at least n + 1 ) will also be considered for the analysis. Among these TCR clones, average clonality within control tumors is compared to MCV-treated tumors. TCR-specific T-cells that are expanded by treatment with MCV in vivo may include a mixture of regulatory cell types, including T helper type 2 (Th2) (secreting IL-4), Th3 [secreting transforming growth factor (TGF)~P], and Tr1 (secreting IL-10) cells, each of which contributes to an anticancer effect.Example 14. Determining Apoptosis and Necrosis In Vitro.

[0162] In vitro experiments with cancerous cell lines, were performed. Necrosis was measured with an Annexin V assay. Annexin V is a 35-36 kDa, Ca2+-dependent phospholipid-binding protein. It has a high affinity for the anionic phospholipid phosphatidylserine (PS). During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and, therefore, can be used as a probe for detecting apoptotic cells. Cells treated with MCV resulted in the greatest apoptosis (Fig. 30A). Staurosporine (stauro) was used as a control.

[0163] Necrosis was also measured. Cells treated with MCV resulted in increased levels of necrosis compared to vehicle control (PBS) treated cells (Fig. 30B).Doxorubicin (doxo) was used as a positive control.

Claims

CLAIMS1 . A method of treating cancer comprising: systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof.

2. The method of claim 1 , wherein the cancer is pancreatic ductal adenocarcinoma (PDAC) or breast cancer.

3. The method of claim 2, wherein the breast cancer is triple negative breast cancer (TNBC).

4. The method of claim 1 , wherein the one or more microbe-derived vaccines is a meningococcal vaccine, a Haemophilus influenzae vaccine, or a pneumococcal vaccine.

5. The method of claim 1 , wherein the one or more microbe-derived vaccines is two or more microbe-derived vaccines.

6. The method of claim 1 , wherein the therapeutically effective dose comprises one or more doses of the one or more microbe-derived vaccine.

7. The method of claim 6, wherein at least one of the one or more doses is administered before the subject is identified as having the cancer.

8. The method of claim 1 , further comprising administering at least one additional anticancer treatment.

9. The method of claim 8, wherein the at least one additional anticancer treatment is surgical therapy, chemotherapy, radiation therapy, hormonal therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radioablation, or a biological therapy.

10. The method of claim 8, wherein the at least one additional anticancer treatment is an immune checkpoint inhibitor.1 1 .The method of claim 10, wherein the at least one checkpoint inhibitor is an inhibitor of Cytotoxic T-lymphocyte antigen 4 (CTLA-4).

12. A method of reducing the progression of a cancer comprising: systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to a subject in need thereof.

13. The method of claim 12, wherein the cancer is a pancreatic ductal adenocarcinoma (PDAC) or breast cancer.

14. The method of claim 13, wherein the breast cancer is triple negative breast cancer (TNBC).

15. A method of increasing the amount of resident memory T-cells (Trms) in a tumor of a subject identified as having a cancer, the method comprising systemically administering a therapeutically effective dose of one or more microbe-derived vaccines to the subject having the tumor, wherein the amount of Trms is compared to a reference level of Trms in a tumor of the same cancer.

16. The method of claim 15, wherein the cancer is a pancreatic ductal adenocarcinoma (PDAC) or breast cancer.

17. The method of claim 16, wherein the breast cancer is triple negative breast cancer (TNBC).

18. The method of claim 15, further comprising identifying the subject as having the cancer.

19. The method of claim 15, further comprising determining the amount of Trms in the tumor after administering the therapeutically effective dose of one or more microbe- derived vaccines to the subject having the tumor.

20. The method of claim 15, further comprising determining the reference level of T rms in a tumor of the same cancer.

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