Intratumoral yeast vaccine

Abstract

The present invention relates to a composition for injection into a solid tumor and a method for treating a solid tumor comprising intratumoral injection of an effective amount of a composition comprising an effective amount of a yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 317,628, filed March 8, 2022. The entire disclosure of U.S. Provisional Patent Application No. 63 / 317,628 is incorporated herein by reference in its entirety. [Background technology]

[0002] Cancer is the leading cause of death worldwide, and the development of effective cancer treatments continues to be one of the most active areas of research and clinical development.Although various innovative approaches have been proposed to treat and prevent cancer, many cancers continue to have high mortality rates and may be difficult to treat or relatively unresponsive to conventional treatments.Therefore, there remains a need for effective cancer treatments. Summary of the Invention [Means for solving the problem]

[0003] One embodiment relates to a composition for injection into a solid tumor, the composition comprising an effective amount of yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

[0004] Another embodiment relates to a method of treating a solid tumor comprising intratumoral injection of an effective amount of a composition comprising yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

[0005] In one aspect of any of the embodiments, the yeast is Saccharomyces cerevisiae.

[0006] In yet another aspect of any of the embodiments, the yeast is genetically engineered to express a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

[0007] In one aspect of any of the embodiments, the Ox40 or CD40 receptor agonist is a monoclonal antibody or a receptor ligand.

[0008] In yet another aspect of any of the embodiments, the tumor is mucosal, melanoma, lung, or colorectal.

[0009] In yet another aspect of any of the embodiments, the tumor is metastatic.

[0010] In another aspect of any of the embodiments, the yeast is genetically engineered to express Protein G on the surface of the yeast.

[0011] In another aspect of any of the embodiments, the Ox40 or CD40 receptor agonist is an Fc agonist fusion protein, and the Fc agonist fusion protein is conjugated to the yeast prior to administration.

[0012] In yet another aspect of any of the embodiments, the yeast is genetically engineered to express an immunostimulatory protein.

[0013] In yet another aspect of any of the embodiments, the IL-15 is nogapendekin-alfa-invaxcept (an NAI also referred to herein as "N-803"). [Brief description of the drawings]

[0014] [Figure 1-1]Figure 1 shows that anti-CTLA-4 antibody improves the anti-tumor activity of (yeast + aOX40) therapy. Figures 1A and 1B show tumor volumes of treated (Figures 1A-1D) and untreated (Figures 1E-1H) tumors up to day 14 post-challenge (pc). Statistics, one-way ANOVA at day 10. Figures 1I-1J show Kaplan-Meier survival analysis up to day 28 (pc). Statistics, one-way ANOVA (tumor volume) or log-rank (KM); ***, p<0.001; ****, p<0.0001. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 1-4] This is a continuation of Figure 1-3. [Figure 1-5] This is a continuation of Figure 1-4. [Figure 1-6] This is a continuation of Figure 1-5. [Diagram 2] Shows that isotype control antibody is less effective than anti-CTLA4 and anti-OX40 in a bilateral tumor model. KM survival curves are shown for treated (FIG. 2A) and untreated (FIG. 2B) tumors. Statistics for FIG. 2A: Log-rank analysis of each group versus PBS; ***p<0.001. "170" is intact yeast expressing the B16F10 "polytope", but the polytope only has one actual epitope (p11) and is not required for efficacy. [Figure 3A]Figure 3 shows that intratumoral (IT) injection of intact or lysed neoepitope yeast + αCTLA4 / αOX40 eradicates B16F10 tumors and confers tumor protection upon tumor rechallenge. Figure 3A shows the tumor growth rate of primary tumors, with complete response rates of 8 / 9 and 7 / 8 mice for intact and lysed yeast, respectively. Figure 3B shows that complete responders were rechallenged with 130,000 B16F10 tumor cells on day 43 and growth was monitored for an additional 94 days. An additional exploratory group of 5 naïve mice was vaccinated subcutaneously with 2.5YU intact and 2.5YU pressure-homogenized pTK170 yeast and 25μg of each antibody was delivered intraperitoneally at the same time (grey). Statistics by two-way ANOVA with Dunnett's multiple comparisons: FIG. 3A, p<0.0001, intact and lysate groups compared to PBS from day 8 onwards; FIG. 3B, intact and lysate groups compared to naive from day 21 onwards. [Figure 3B] Figure 3 shows that intratumoral (IT) injection of intact or lysed neoepitope yeast + αCTLA4 / αOX40 eradicates B16F10 tumors and confers tumor protection upon tumor rechallenge. Figure 3A shows the tumor growth rate of primary tumors, with complete response rates of 8 / 9 and 7 / 8 mice for intact and lysed yeast, respectively. Figure 3B shows that complete responders were rechallenged with 130,000 B16F10 tumor cells on day 43 and growth was monitored for an additional 94 days. An additional exploratory group of 5 naïve mice was vaccinated subcutaneously with 2.5YU intact and 2.5YU pressure-homogenized pTK170 yeast and 25μg of each antibody was delivered intraperitoneally at the same time (grey). Statistics by two-way ANOVA with Dunnett's multiple comparisons: FIG. 3A, p<0.0001, intact and lysate groups compared to PBS from day 8 onwards; FIG. 3B, intact and lysate groups compared to naive from day 21 onwards. [Figure 4A] Figure 4 shows the effect of αOX40 and αCTLA4 antibody dose on anti-tumor activity in the B16F10 triple therapy model. Primary tumor growth rates are shown up to day 8 (Figure 4A) and day 17 (Figure 4B) post-treatment. Statistics by two-way ANOVA with Dunnett's multiple comparisons compared to PBS; significance applies to all lysate cohorts. ***, p<0.001; ****, p<0.0001. [Figure 4B] Figure 4 shows the effect of αOX40 and αCTLA4 antibody dose on anti-tumor activity in the B16F10 triple therapy model. Primary tumor growth rates are shown up to day 8 (Figure 4A) and day 17 (Figure 4B) post-treatment. Statistics by two-way ANOVA with Dunnett's multiple comparisons compared to PBS; significance applies to all lysate cohorts. ***, p<0.001; ****, p<0.0001. [Diagram 5] Figure 5 shows the effect of αOX40 and αCTLA4 antibody dose on anti-tumor activity in the B16F10 triple therapy model. Figure 5A shows the tumor growth rate (volume) up to day 52 after treatment. Figure 5B shows the Kaplan-Meier survival plot for the same period. [Figure 6-1] Intratumoral injection of yeast displaying Fc-OX40L is shown. Figures 6A-6B: Yeast was engineered to express surface-displayed protein G, in this case Fc-OX40L, which binds any Fc-containing protein. Figure 6A, schematic of strain; Figure 6B, flow cytometry analysis. Figure 6C shows the intratumoral injection regimen and treatment groups. The effect of treatment on tumor growth and survival of treated (Figures 6D-6J) and untreated (Figures 6K-6Q) tumors is shown. For both Figures 6D-6I and 6K-6P, spider plots show tumor growth for individual mice, and Kaplan-Meier curves (Figures 6J and 6Q) show survival. Statistics by log rank, **p<0.01 for the comparisons shown. EBY100, parent yeast strain; 424, empty yeast expression plasmid; Mox40L-Fc, murine OX40L fused to IgG1 Fc. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 6-3] This is a continuation of Figure 6-2. [Figure 6-4] This is a continuation of Figure 6-3. [Figure 6-5] This is a continuation of Figure 6-4. [Figure 6-6] This is a continuation of Figure 6-5. [Figure 6-7] This is a continuation of Figure 6-6. [Figure 6-8] This is a continuation of Figure 6-7. [Figure 6-9]This is a continuation of Figure 6-8. [Figure 6-10] This is a continuation of Figure 6-9. [Figure 6-11] Continuation of Figure 6-10. [Figure 7] Figure 7 shows that N803 can replace αOX40 in a B16F10 intratumoral injection assay.Tumor growth of injected (primary) tumors was monitored for 38 days (Figure 7A) or 68 days (Figure 7B). [Figure 8-1] Figure 8A-8E: Primary tumor growth was monitored for 42 days. On day 45, complete responders were re-challenged intradermally in the contralateral (left) flank with 300,000 MC38 tumor cells and tumor volume (Figure 8F-8J) and survival (Figure 8K) were monitored for an additional 48 days. W303a pL, pressure homogenized yeast null lysate. Arrows indicate it injection. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 8-3] This is a continuation of Figure 8-2. [Figure 8-4] This is a continuation of Figure 8-3. [Figure 8-5] This is a continuation of Figure 8-4. [Figure 8-6] This is a continuation of Figure 8-5. [Figure 8-7] This is a continuation of Figure 8-6. [Figure 9A] Titration of anti-PDL1 / anti-OX40 antibodies for intratumoral treatment of MC38 tumors. Female C57BL / 6 mice were implanted intradermally (see schematic FIG. 9A). Survival was monitored for 48 days (FIG. 9B). W303a=yeast null; pL=pressure lysate. Statistics: Mantel-Cox test compared to PBS*;^compared to group with same dose of antibody but lacking yeast pL injection. it injections are indicated by arrows. [Figure 9B]Titration of anti-PDL1 / anti-OX40 antibodies for intratumoral treatment of MC38 tumors. Female C57BL / 6 mice were implanted intradermally (see schematic FIG. 9A). Survival was monitored for 48 days (FIG. 9B). W303a=yeast null; pL=pressure lysate. Statistics: Mantel-Cox test compared to PBS*;^compared to group with same dose of antibody but lacking yeast pL injection. it injections are indicated by arrows. [Figure 10-1] Potent therapeutic effect of aPDL1 / aOX40 + yeast lysate intratumoral therapy in a bilateral MC38 implantation model. Female C57BL / 6 mice were implanted intradermally (see FIG. 10A). The impaired growth of treated (FIG. 10B-10F) and untreated (FIG. 10G-10K) tumors is shown, and the cure rates are shown. (FIG. 10L) Kaplan-Meier curves showing overall survival up to day 58 after challenge (population data, treated and untreated tumors). Mortality events (sacrifice) were recorded when a single tumor reached 1000 mm3 or when the combined treated and untreated tumor volume reached 1500 mm3. Statistics by Mantel-Cox test for treatment groups compared to PBS: **, P<0.01; ****, P<0.0001. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-2. [Figure 10-4] This is a continuation of Figure 10-3. [Figure 10-5] This is a continuation of Figure 10-4. [Figure 10-6] Continuation of Figure 10-5. [Figure 10-7] Continuation of Figure 10-6. [Figure 10-8] This is a continuation of Figure 10-7. [Figure 11A] Figure 11A shows an experimental schematic of the intratumoral (IT) injection assay model for evaluation of anti-CTLA4 ("a-CTLA4-7" in Figure 11B), anti-PD-L1 ("a-PD-L-1" in Figure 11B), N-803 and yeast ("W303α" in Figure 11B) in a two-tumor IT B16.F10 mouse model. Figure 11B shows a chart of the amount and route of administration of each treatment. [Figure 11B]Figure 11A shows an experimental schematic of the intratumoral (IT) injection assay model for evaluation of anti-CTLA4 ("a-CTLA4-7" in Figure 11B), anti-PD-L1 ("a-PD-L-1" in Figure 11B), N-803 and yeast ("W303α" in Figure 11B) in a two-tumor IT B16.F10 mouse model. Figure 11B shows a chart of the amount and route of administration of each treatment. [Figure 12-1] Using the IT assay model disclosed in Figures 11A-11B, we show that tumors were eliminated following injection of 50 μg of anti-CTLA4, anti-PD-L1, N-803, and yeast. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-2. [Figure 12-4] This is a continuation of Figure 12-3. [Figure 13-1] Using the IT assay model disclosed in Figures 11A-11B, we show that IT injection of anti-CTLA4, anti-PD-L1, N-803, and yeast delayed tumor growth of non-injected distant tumors. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 13-3] This is a continuation of Figure 13-2. [Figure 13-4] This is a continuation of Figure 13-3. [Figure 14A] FIG. 1 shows a comparison of intact yeast (INT) and clarified lysate (CL: (INT yeast was pressure lysed, centrifuged, and filtered)) yeast preparations in a two-tumor B16.F10 melanoma mouse model. [Figure 14B] FIG. 1 shows a comparison of intact yeast (INT) and clarified lysate (CL: (INT yeast was pressure lysed, centrifuged, and filtered)) yeast preparations in a two-tumor B16.F10 melanoma mouse model. [Figure 14C] FIG. 1 shows a comparison of intact yeast (INT) and clarified lysate (CL: (INT yeast was pressure lysed, centrifuged, and filtered)) yeast preparations in a two-tumor B16.F10 melanoma mouse model. [Figure 14D]FIG. 1 shows a comparison of intact yeast (INT) and clarified lysate (CL: (INT yeast was pressure lysed, centrifuged, and filtered)) yeast preparations in a two-tumor B16.F10 melanoma mouse model. [Figure 15-1] Comparison of IT (also referred to as "it") injection on tumor growth inhibition compared to intraperitoneal (IP or ip) and / or subcutaneous (SC or sc) injection routes of administration: PBS (control - top line); 50μg α-PD-L1 / α-CTLA (ip) + 5YU W303INT (sc) + 2μg N-803 (sc) (middle line); and 50μg α-PD-L1 / α-CTLA + 5YU W303INT + 2μg N-803 (it). [Figure 15-2] This is a continuation of Figure 15-1. [Figure 16] Shown are the results of IT or IP / SC injection of anti-CTLA4 / anti-PD-L1 / N-803 / yeast on mouse body weight. [Figure 17] FIG. 1 shows an experimental schematic for the evaluation of N-803 dose schedules in combination IT therapy in a two-tumor B16.F10 melanoma mouse model. [Figure 18] The results of co-injection of anti-CTL4 / yeast and N-803 given every 2 days for 3 times are shown for inhibition of tumor growth of injected tumors. [Figure 19] 1 shows a comparison of 2-day and 4-day dosing intervals of N-803 on the growth of injected tumors. [Figure 20] The results of co-injection of anti-CTL4 / yeast and N-803 given every 2 days for 3 times on inhibition of tumor growth of non-injected tumors are shown. [Figure 21] A comparison of 2-day and 4-day N-803 dose intervals on non-tumor growth is shown. [Figure 22] [Figure 22A] Experimental schematic assay to evaluate IT injected N-803 / anti-PD-L1 + yeast INT or CL formulations in a two-tumor MC38 colorectal cancer model. [Figure 22B] Chart of amounts and formulations ("vac info") used for each assay group. [Figure 23-1]Results are shown for growth delay of injected tumors using the assays and formulations shown in Figures 22A and 22B. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 23-3] Continuation of Figure 23-2. [Figure 24-1] Results are shown for growth delay of non-injected tumors using the assays and formulations shown in Figures 22A and 22B. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 24-3] Continuation of Figure 24-2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by those of ordinary skill in the art. In general, nomenclature used in connection with and in the arts of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry is that well known and commonly used in the art.

[0016] All publications, patents, and published patent applications referenced in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0017] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" refers to the inclusion of a specified integer (or component) or group of integers (or components) but not to the exclusion of any other integer (or component) or group of integers (or components).

[0018] The singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0019] "Including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0020] A pharma- ceutically acceptable carrier means a non-toxic carrier that may be administered to a patient together with a composition described herein and that does not destroy the pharmacological activity of the active agent within the composition. An "excipient" means an additive in a formulation or composition that is not a pharma- ceutically active ingredient.

[0021] "Peptide" and "polypeptide" are used interchangeably herein to refer to a polymer composed of amino acid residues. As used herein, "amino acid residue" refers to any naturally occurring amino acid (L or D form), a non-naturally occurring amino acid, or an amino acid mimetic (such as a peptide monomer).

[0022] "Identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences means two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window. For purposes of this disclosure, the degree of amino acid or nucleic acid sequence identity is determined using the BLAST algorithm described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. This algorithm identifies high scoring sequence pairs (HSPSs) by identifying short words of length W in a query sequence that match or meet some positive threshold score when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., (1990) J. Mol. Biol. 215:403-10). The initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended along each sequence in both directions for as far as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below 0 due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is not reached. For determining percent identity of amino acid sequences, the BLASTP settings are: word length (W), 3; expectation (E), 10; and the BLOSUM62 score matrix. For analysis of nucleic acid sequences, the BLASTN program settings are: word length (W), 11; expectation (E), 10; M=5; N=-4; and comparison of both strands.The BLASTN program (protein sequence against nucleotide sequence database query) uses a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0023] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87). The smallest sum probability (P(N)) provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability is less than about 0.01 in a comparison of the test nucleic acid to the reference nucleic acid.

[0024] Unless otherwise specified, each embodiment disclosed herein can be used alone or in combination with any one or more of the other embodiments herein.

[0025] Disclosed herein are compositions for injection into solid tumors and methods for treating solid tumors.

[0026] One embodiment relates to a composition for injection into a solid tumor, the composition comprising an effective amount of yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

[0027] One embodiment relates to a method of treating a solid tumor comprising intratumoral injection of an effective amount of a composition comprising yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

[0028] Solid tumors are heterogeneous aggregates of many cell types, including cancer cells, cancer stem cells, connective tissue cells, and immune cells. All of these cells can communicate with each other through specific junctions (tight junctions and gap junctions) that maintain and regulate the complete tumor microenvironment. Several scientific studies have shown that these different genetically engineered tumors with acquired resistance may control complex cell-cell interactions and abnormal signaling pathways that lead to genomic instability. Tumor heterogeneity indicates poor prognosis for survival. Examples of solid tumor cancers include mucosal, melanoma, lung, colorectal, breast, bladder, and prostate. In one aspect, the tumor is mucosal, melanoma, lung, or colorectal. In yet another aspect, the tumor is metastatic.

[0029] "TARMOGEN®" (GlobeImmune, Inc., Louisville, Colorado) generally refers to a yeast vehicle expressing one or more heterologous antigens extracellularly (on its surface), intracellularly (inside or in the cytoplasm), or both extracellularly and intracellularly. TARMOGEN® has been generally described (see, e.g., U.S. Pat. No. 5,830,463). Certain yeast-based immunotherapy compositions, as well as methods of making and generally using the same, are also described in detail in, for example, U.S. Pat. Nos. 5,830,463, 7,083,787, 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and Bernstein et al., Vaccine 2008 Jan 24;26(4):509-21, each of which is incorporated herein by reference in its entirety.

[0030] The composition includes yeast, yeast vehicle and / or yeast lysate. In one aspect, the yeast lysate is prepared from yeast and is devoid of yeast membrane and yeast cell wall. Such yeast lysate is prepared from lysed yeast, i.e., yeast whose cell wall and membrane have been disrupted, exposing the yeast cell contents to the remainder of the composition. The yeast lysate can be prepared from inactivated yeast, such as heat-inactivated yeast, or from live yeast. The yeast can contain disease-associated antigens expressed inside the yeast from a plasmid or integrated chromosomal allele. For example, yeast can be lysed by glass bead bursting by mixing with PBS and 500 μL of acid-washed 0.2 μm glass beads in a total volume of 1.5 mL and shaking the mixture vigorously in a mechanical stirrer until the cells burst, e.g., >97% of the cells burst. Alternatively, yeast can be lysed by other methods including high pressure homogenization, sonication, and electrical, physical, chemical and enzymatic techniques. (See, e.g., U.S. Patent Application Serial No. 17 / 047,134).

[0031] In any of the yeast-based compositions used in the present invention, the following aspects related to yeast vehicles are included in the present invention. According to the present invention, a yeast vehicle is any yeast cell (e.g., whole or intact cell) or its derivative (see below) that can be used in conjunction with one or more antigens, immunogenic domains thereof or epitopes thereof in the therapeutic composition of the present invention. Thus, a yeast vehicle can include, but is not limited to, live intact yeast microorganisms (i.e., yeast cells with all components including cell wall), killed (dead) or inactivated intact yeast microorganisms, or yeast spheroplasts (i.e., yeast cells lacking cell wall), yeast cytoplasts (i.e., yeast cells lacking cell wall and nucleus), yeast ghosts (i.e., yeast cells lacking cell wall, nucleus and cytoplasm), intracellular yeast membrane extracts or fractions thereof (also called yeast membrane particles, formerly called intracellular yeast particles), any other yeast particles, or derivatives thereof, including yeast cell wall preparations.

[0032] Yeast spheroplasts are typically produced by enzymatic digestion of the yeast cell wall, as described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674, which is incorporated herein by reference in its entirety.

[0033] Yeast cytoplasts are typically produced by enucleation of yeast cells, such as described, for example, in Coon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55, which is incorporated herein by reference in its entirety.

[0034] Yeast ghosts are typically produced by resealing permeabilized or lysed cells and may, but need not, contain at least some of the organelles of that cell. Such methods are described, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporated herein by reference in its entirety.

[0035] Yeast membrane particles (intracellular yeast membrane extract or fraction thereof) refers to yeast membranes that lack a native nucleus or cytoplasm. The particles may be of any size, including the size of a native yeast membrane to microparticles produced by sonication or other membrane disruption methods known to those skilled in the art, followed by resealing. Methods for producing intracellular yeast membrane extracts are described, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194, 662-674. Fractions of yeast membrane particles that contain yeast membrane portions, and antigens or other proteins of interest can also be used if the antigens or other proteins were recombinantly expressed by yeast prior to preparation of the yeast membrane particles. The antigens or other proteins of interest can be carried inside the membrane, on any surface of the membrane, or a combination thereof (i.e., the protein can be both inside and outside the membrane and / or can penetrate the membrane of the yeast membrane particle). In one embodiment, the yeast membrane particle is a recombinant yeast membrane particle, which can be an intact, disrupted, or disrupted and resealed yeast membrane that contains at least one desired antigen or other protein of interest on the surface of the membrane or at least partially embedded within the membrane.

[0036] An example of a yeast cell wall preparation is an isolated yeast cell wall carrying an antigen on its surface or at least partially embedded within the cell wall, such that the yeast cell wall preparation stimulates a desired immune response against a disease target when administered to an animal.

[0037] In the case of compositions containing lysed yeast cells, these compositions can then be further processed to remove yeast membranes and yeast cell walls by any suitable method to produce a yeast lysate lacking yeast membranes and yeast cell walls. For example, yeast membranes and yeast cell walls can be removed from the lysed yeast by centrifugation to produce a lysate (supernatant) free of cell walls and membranes, such as by centrifugation of the lysed yeast at 16,000 rpm for 5 minutes at 25° C. Alternatively, the lysate can be removed of cell walls and membrane debris after rupture by means other than centrifugation. Filtration or treatment of cells, for example with conA beads, is an alternative method. In an exemplary method, the pressure lysate is centrifuged to pellet / remove insoluble particles, and then the supernatant is filtered for sterilization. When referring to removing yeast membranes and yeast cell walls or to a lysate lacking yeast membranes and yeast cell walls, it will be recognized that a suitable process for removing material may not remove 100% of the yeast membranes and yeast cell walls from the lysate. Thus, in some cases, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of the yeast membrane and / or yeast cell wall is removed.

[0038] The compositions of the invention include at least one antigen heterologous to the yeast (this term includes the immunogenic domain of the antigen) in addition to a yeast lysate lacking yeast membranes and yeast cell walls. The heterologous antigen may be expressed by the yeast, such as before lysing the yeast when the yeast vehicle is a yeast lysate, or the antigen may be combined with the yeast before or after lysis and before or after removing the yeast membranes and yeast cell walls from the lysate. In some embodiments, the antigen is provided as a fusion protein that may include two or more antigens. The antigen may, in some embodiments, elicit an antigen-specific immune response (e.g., humoral and / or cell-mediated immune response) against the same or similar antigen encountered by elements of the immune system (e.g., T cells, antibodies). In one aspect, the fusion protein may include two or more immunogenic domains of one or more antigens, or two or more epitopes of one or more antigens. In one aspect, the yeast is engineered to express a tumor-associated antigen, a tumor-specific antigen, or a neo-epitope.

[0039] The present invention includes the use of at least one "composition" (which phrase may be used interchangeably with "yeast-based immunotherapy composition", "yeast-based immunotherapy product", "yeast-based immunotherapy composition", "yeast-based composition", "yeast-based immunotherapy" or "yeast-based vaccine"), which may be yeast, a yeast vehicle, or a yeast lysate lacking yeast membranes and yeast cell walls, alone or in combination with an intact yeast-based immunotherapy composition such as TARMOGEN®. The yeast-based immunotherapy composition elicits an immune response sufficient to achieve at least one therapeutic benefit in a subject. More specifically, a yeast-based immunotherapy composition is a composition comprising yeast, a yeast vehicle, or a yeast lysate component, alone or in combination with an intact yeast-based immunotherapy composition, which is capable of eliciting or inducing an immune response, such as a cellular immune response, including but not limited to a T cell-mediated cellular immune response. In one aspect, the yeast-based immunotherapy composition useful in the present invention is capable of inducing a CD8+ and / or CD4+ T cell-mediated immune response, and in one aspect, a CD8+ and CD4+ T cell-mediated immune response. In some cases, the yeast-based immunotherapy composition can induce a humoral immune response.

[0040] Yeast lysates can be made from intact yeast-based immunotherapeutic compositions (i.e., TARMOGEN®). Additionally, such intact yeast-based immunotherapeutic compositions can be combined with yeast lysate-based compositions. Such intact yeast-based immunotherapeutic compositions generally include a yeast vehicle (in this case, not including yeast lysate) and an antigen heterologous to the yeast.

[0041] Such intact yeast-based immunotherapy compositions, as well as methods for making and generally using the same, have been described in detail, for example, in U.S. Patent No. 5,830,463, U.S. Patent No. 7,083,787, U.S. Patent No. 7,465,454, U.S. Patent Application Publication No. 2007-0224208, U.S. Patent Application Publication No. 2008-0003239, as well as Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., Cancer Research 64:5084-5088 (2004), and Bernstein et al., Vaccine 2008 Jan 24;26(4):509-21, each of which is incorporated herein by reference in its entirety. These yeast-based immunotherapy products have been shown to elicit immune responses, including cellular and humoral immune responses. Yeast-based immunotherapy products can kill target cells expressing various antigens in vivo in various animal species, and can kill through antigen-specific, CD4+ and CD8+ mediated immune responses. Further studies have shown that yeast is potently phagocytosed by dendritic cells, directly activated, and then presents yeast-associated proteins to CD4+ and CD8+ T cells in a highly efficient manner. See, for example, Stubbs et al. Nature Med. 5:625-629 (2001) and U.S. Patent No. 7,083,787.

[0042] Any yeast strain can be used to generate the yeast vehicle (for production of a yeast lysate or for use in combination with a yeast lysate). In one embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the yeast is Pichia Pastoris.

[0043] The yeast vehicle of the present invention can fuse with the cell type to which the yeast vehicle and antigen / agent are to be delivered, such as a dendritic cell or macrophage, thereby resulting in particularly efficient delivery of the yeast vehicle and, in many embodiments, the antigen or other agent to the cell type. As used herein, fusion of the yeast vehicle with the target cell type refers to the ability of the yeast cell membrane or particles thereof to fuse with the membrane of the target cell type (e.g., a dendritic cell or macrophage), resulting in syncytium formation. As used herein, a syncytium is a multinucleated mass of protoplasm produced by the coalescence of cells. It has been shown that many viral surface proteins (including those of immunodeficiency viruses such as HIV, influenza virus, poliovirus, and adenovirus) and other fusogens (such as those involved in fusion between eggs and sperm) can result in fusion between two membranes (i.e., between a virus and a mammalian cell membrane or between mammalian cell membranes). For example, yeast vehicles that produce HIV gp120 / gp41 heterologous antigens on their surface can fuse with CD4+ T lymphocytes. However, it is noted that incorporating targeting moieties into yeast vehicles, although desirable under some circumstances, is not necessary. In the case of yeast vehicles that express antigens extracellularly, this may be an additional advantage of the yeast vehicles of the present invention. In general, yeast vehicles useful in the present invention are easily taken up by dendritic cells (and other cells such as macrophages).

[0044] Expression of antigens or other proteins in yeast or yeast vehicles of the present invention is achieved using techniques known to those skilled in the art. Briefly, a nucleic acid molecule encoding at least one desired antigen or other protein is inserted into an expression vector such that the nucleic acid molecule is operably linked to a transcription control sequence to achieve either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell. The nucleic acid molecule encoding one or more antigens and / or other proteins may be present on one or more expression vectors operably linked to one or more expression control sequences. Particularly important expression control sequences are those that control transcription initiation, such as promoters and upstream activating sequences. Any suitable yeast promoter may be used in the present invention, and a variety of such promoters are known to those skilled in the art.

[0045] In another aspect, the antigen and / or other agent is physically bound to the yeast or yeast vehicle. The physical binding of the antigen and / or other agent to the yeast or yeast vehicle can be achieved by any method suitable in the art, including covalent and non-covalent association methods, including but not limited to chemically cross-linking the antigen and / or other agent to the outer surface of the yeast vehicle, or biologically linking the antigen and / or other agent to the outer surface of the yeast vehicle (e.g., by using an antibody or other binding partner). Chemical cross-linking can be achieved by methods including, for example, glutaraldehyde binding, photoaffinity labeling, treatment with carbodiimide, treatment with chemicals that can link disulfide bonds, and treatment with other cross-linking chemicals standard in the art. Alternatively, a chemical can be contacted with the yeast or yeast vehicle that changes the charge of the lipid bilayer or cell wall composition of the yeast membrane, such that the outer surface of the yeast is more likely to fuse or bind to antigens and / or other agents with certain charge characteristics. Targeting agents such as antibodies, binding peptides, soluble receptors, and other ligands may also be incorporated into the antigen as fusion proteins or otherwise associated with the antigen for binding of the antigen to the yeast or yeast vehicle.

[0046] In one aspect, the compositions disclosed herein comprise a checkpoint inhibitor. Preferred checkpoint inhibitors include anti-CTLA-4 antibodies and anti-PDL1 antibodies.

[0047] In one aspect, the compositions disclosed herein comprise immunostimulatory proteins, including OX40 receptor agonists, CD40 receptor agonists, and IL-15 superagonists.

[0048] OX40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and regulates multiple T cell functions. Cell surface expression of OX40 is upregulated after T cell activation; upon binding to OX40 ligand (OX40L), it provides a costimulatory signal and inhibits CD4+ T cell proliferation in preclinical studies. + and CD8 + Increases activation of T effector cells (Aspeslagh, S, et al. Rationale for anti-OX40 cancer immunotherapy. Eur J Cancer 2016;52:50-66; Jensen, SM., et al., Signaling through OX40 enhances antitumor immunity. Semin Oncol. 2010;37:524-32; and Piconese S, et al., OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection. J Exp med 2008;205:825-39). OX40 can also inhibit regulatory T cell-mediated suppression, block the generation of regulatory T cells, and result in enhanced T effector cell activity (Piconese S, et al, 2008). Agonistic OX40 antibodies can induce survival and proliferation of tumor antigen-specific CD4 T cells.

[0049] CD40 (cluster of differentiation 40) is a costimulatory protein found on antigen-presenting cells and is required for their activation. CD40L (CD40 ligand) binds to CD40 on antigen-presenting cells.

[0050] In one aspect, the OX40 or CD40 receptor agonist is an Fc agonist fusion protein. In a preferred embodiment, the Fc agonist fusion protein is bound to the yeast prior to administration of the composition.

[0051] N-803 (also known as ALT-803 or nogapendekin-alfa-invaxcept (NAI)) is an IL-15-based immunostimulatory protein complex containing two protein subunits of a human IL-15 variant associated with high affinity for dimeric human IL-15 receptor alpha (IL-15Rα) sushi domain / human IgG1 Fc fusion protein. The IL-15 variant is the mature human IL-15 cytokine sequence (114 amino acids) with an asparagine to aspartic acid substitution at position 72 (N72D). The human IL-15Rα sushi domain / human Fc fusion protein contains the sushi domain of the human IL-15 receptor alpha subunit (IL-15Rα) (amino acids 1-65 of the mature human IL-15Rα protein) linked to a human IgG1 CH2-CH3 region that contains the Fc domain (232 amino acids). With the exception of the N72D substitution, all protein sequences are human. N-803 is an IL-15 superagonist.

[0052] In one embodiment, the yeast is engineered to express Protein G on the surface of the yeast. In yet another embodiment, the yeast is engineered to express an immunostimulatory protein disclosed herein, such as OX40, OX40L, CD40, and CD40L. In yet another embodiment, the yeast expressing Protein G is coupled to an immunostimulatory protein.

[0053] According to the methods provided herein, a subject is administered an effective, pharma- ceutical, or therapeutically effective amount of a composition or a component of a composition provided herein. The terms effective, therapeutic, pharma- ceutical, and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., reduction in tumor size, elimination of tumor; killing cells, and / or preventing the growth of new cells). Effective amounts and schedules for administering the compositions disclosed herein can be empirically determined by one of skill in the art. The dosage range of administration is large enough to produce the desired effect (e.g., reduction or delay) in which one or more symptoms of a disease or disorder are affected. The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. In general, dosage will vary depending on age, medical condition, sex, type of disease, extent of disease or disorder, route of administration, or whether other drugs are included in the dosing regimen, and can be determined by one of skill in the art. Dosage can be adjusted by the individual physician in case of contraindications. Dosage can vary and can be administered daily, in one or multiple doses, for one or several days. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceuticals. For example, for a given parameter, an effective amount indicates a decrease or reduction of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect compared to a control.The exact dosage or formulation will vary depending on the purpose of treatment, and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Editor (2012), and Pickar, Dosage Calculations (1999)).

[0054] In the context of the compositions disclosed herein, generally, a suitable single dose is one that, when administered one or more times over a suitable period of time, can effectively provide a given tumor with the composition of the invention in an amount effective to elicit an antigen-specific immune response. For example, in one embodiment, a single dose of a composition of the invention is about 1×10 per kg body weight of the organism to which the composition is administered. 5 ~Approx. 5×10 7 In one embodiment, a single dose of the yeast vehicle of the present invention is 0.1×10 yeast cell equivalents. 6 Increment of cells (i.e., 1.1 x 10 6 , 1.2×10 6 , 1.3×10 6 ...), including any intermediate doses, approximately 0.1 YU (1 × 10 6 cells)~about 200Y.U.(2×10 9 In one aspect, the dose comprises a dose of 1 Y.U. to 80 Y.U., in one aspect, a dose of 5 Y.U. to 40 Y.U. In one embodiment, the dose is administered via intratumoral injection three times, every two days.

[0055] A "booster" or "boost" of the compositions disclosed herein can be administered.

[0056] Terms such as subject, patient, individual, etc. are not intended to be limiting and can generally be interchanged. As used throughout, a subject can be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not indicate a specific age or sex. Thus, adult and neonatal subjects, whether male or female, are intended to be covered. As used herein, patient, individual, and subject can be used interchangeably, and these terms are not intended to be limiting. That is, an individual described as a patient does not necessarily have a given disease, but may simply be seeking medical advice. The term patient or subject includes human and veterinary subjects.

[0057] References to "therapeutic" herein should be considered in their broadest context. "Treatment" does not necessarily mean that a mammal is treated to complete recovery. As used herein, "treat", "treating" and similar words mean to stabilize and / or reduce the symptoms of a disease or condition. In some aspects, the compositions disclosed herein can cure a medical condition or disease apart from the treatment.

[0058] The route and frequency of administration of the therapeutic compositions described herein, as well as the dosage, will vary from individual to individual and from disease to disease, and can be readily established using standard techniques. Preferably, the compositions can be administered by intratumoral injection.

[0059] Compositions suitable for intratumoral injectable use include sterile aqueous solutions (if water soluble) and sterile powders for extemporaneous preparation of sterile injectable solutions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin in the composition.

[0060] Injectable sterile solution can be prepared by incorporating active compound in the required amount in suitable solvent with various other components listed above, and then, if necessary, for example, sterilization by filtration or other suitable means.By incorporating various sterilized active components into sterilized vehicle, dispersion can be prepared, which contains basic dispersion medium and other components required from the components listed above.In the case of sterile powder for preparing injectable sterile solution, the preferred method of preparation includes vacuum drying and freeze-drying technology, which produces powder of active component + any additional desired components from the solution previously sterilized and filtered.

[0061] In conjunction with any of the above methods, the composition can be administered in combination with another drug. In each case, the composition can be administered before, simultaneously, or after administration of the other drug. According to the methods described herein, one or more compounds or compositions can be co-administered with one or more other compounds, such as known chemotherapy and anti-inflammatory drugs (e.g., betamethasone, nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, ibuprofen, naproxen), and / or other suitable drugs. The methods provided can also be combined with other tumor treatments, such as radiation therapy, surgery, hormone therapy, and / or immunotherapy. Thus, the methods provided can further include administering one or more additional therapeutic agents to the subject. Suitable additional therapeutic agents include, but are not limited to, analgesics, anesthetics, analgesics, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastics, allosteric inhibitors, anabolic steroids, antirheumatics, psychotherapeutic agents, nerve blocks, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antivirals, dopaminergic agents, blood system agents, immune agents, muscarinic agents, protease inhibitors, vitamins, growth factors, and hormones. The choice of agent and dosage can be easily determined by those skilled in the art based on the given disease to be treated. Optionally, the additional therapeutic agent is octreotide acetate, interferon, pembrolizumab, glucopyranosyl lipid A, carboplatin, etoposide, or any combination thereof.

[0062] "Co-administered" means simultaneous administration in the same formulation or in two different formulations by the same or different route, or sequential administration by the same or different route. "Sequential" administration means a time difference of seconds, minutes, hours, or days between the administration of two or more separate compounds.

[0063] In some embodiments, it may be beneficial to include one or more excipients in the composition. Those skilled in the art will recognize that the selection of one excipient may affect the selection of the other excipient. For example, the selection of a particular excipient may preclude the use of one or more additional excipients, since the combination of excipients would cause undesirable effects. Those skilled in the art will be able to empirically determine which excipients, if any, to include in the formulations or compositions disclosed herein. Excipients include, but are not limited to, co-solvents, solubilizers, buffers, pH adjusters, bulking agents, surfactants, encapsulating agents, tonicity adjusters, stabilizers, protectants, and viscosity adjusters. In some embodiments, it may be beneficial to include a pharma- ceutical acceptable carrier.

[0064] In some embodiments, it may be beneficial to include a solubilizer. The solubilizer may be useful for enhancing the solubility of any of the components of the formulation or composition, such as the peptides or excipients disclosed herein. The solubilizers described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary solubilizers that may be used. In certain embodiments, the solubilizer includes, but is not limited to, ethyl alcohol, t-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinylpyrrolidone, and pharma- ceutically acceptable salts thereof and / or combinations thereof.

[0065] The pH can be any pH that provides the composition with desirable properties, including, for example, peptide stability, increased peptide retention compared to compositions at other pHs, and improved filtration efficiency.

[0066] In some embodiments, it may be beneficial to include a tonicity adjuster. The tonicity of a liquid composition is an important consideration, for example, when administering the composition to a patient via parenteral administration. Thus, tonicity adjusters may be used to help make the composition suitable for administration. Tonicity adjusters are well known in the art. Thus, the tonicity adjusters described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary tonicity adjusters that may be used. Tonicity adjusters may be ionic or non-ionic, and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars include sugar alcohols, such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

[0067] In some embodiments, it may be beneficial to include a stabilizer, which helps to increase the stability of the peptide in the composition of the invention.

[0068] In some embodiments, it may be beneficial to include a protective agent. A protective agent is an agent that protects a pharma- ceutical active ingredient (e.g., a peptide disclosed herein) from undesirable conditions (e.g., instability caused by freezing or lyophilization, or oxidation). Protective agents include, for example, cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants are useful for preventing loss of potency of an active pharmaceutical ingredient (e.g., a peptide disclosed herein) when the formulation is exposed to a temperature below its freezing point. For example, a cryoprotectant can be included in a reconstituted lyophilized formulation, such that the formulation can be frozen prior to intravenous (IV) administration. Cryoprotectants are well known in the art. Thus, the cryoprotectants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary cryoprotectants that may be used. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Cryoprotectants can include, for example, disaccharides (eg, sucrose, lactose, maltose, and trehalose), polyols (eg, glycerol, mannitol, sorbitol, and dulcitol), glycols (eg, ethylene glycol, polyethylene glycol, propylene glycol).

[0069] Lyoprotectants are useful for stabilizing the components of lyophilized formulations or compositions. For example, the peptides disclosed herein can be lyophilized using a lyoprotectant prior to reconstitution. Lyoprotectants are well known in the art. Thus, the lyoprotectants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary lyoprotectants that may be used. Lyoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizers, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants can be, for example, sugars and polyols, trehalose, sucrose, dextran, with hydroxypropyl-β-cyclodextrin being a non-limiting example of a lyoprotectant.

[0070] Antioxidants are useful for preventing oxidation of the components of the composition. Oxidation can cause drug product aggregation or other adverse effects on drug product purity or its potency. Antioxidants are well known in the art. Thus, the antioxidants described herein are not intended to constitute an exhaustive list, but are merely provided as exemplary antioxidants that can be used. Antioxidants can be, for example, sodium ascorbate, sodium citrate, thiols, metabisulfites, and combinations thereof.

[0071] The present invention also includes kits containing any of the compositions described herein, or any of the individual components of the compositions described herein. The reagents may be in free form or may be immobilized on a substrate, such as a plastic dish, a microarray plate, a test tube, a test stick, etc. The kits may also include appropriate reagents for detection of the reagents and / or for labeling of positive or negative controls, washing solutions, dilution buffers, etc. The kits may also include a set of written instructions for using the kit and interpreting the results. The kits may be prepared and used for any clinical, research, or diagnostic method of the invention.

[0072] Variations, modifications, and other embodiments of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the disclosure, and therefore the following claims are not limited to only the foregoing illustrative description.

[0073] Each of the embodiments described herein may be combined individually or in conjunction with one or more other embodiments of the present invention.

[0074] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the compounds, compositions, and methods of use described herein, which are deemed to be within the scope of the compositions and methods disclosed herein.

[0075] The contents of all references, patents and published patent applications cited throughout this application, as well as any drawings accompanying it, are hereby incorporated by reference in their entirety.

[0076] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention or to represent that the following experiments are complete or the only experiments performed. EXAMPLES

[0077] Example 1 This example shows that anti-CTLA4 antibodies improve the anti-tumor activity of yeast plus anti-OX40 therapy. Using the B16F10 model system, female C57BL / 6 mice were implanted intradermally in the left and right flanks with B16F10 bearing 130,000 B16F10 melanoma tumor cells. When tumors were 5-7 mm in diameter, mice were treated with 5YU of intact yeast expressing the B16F10 neo-epitope (PTK170-a TARMOGEN® expressing the neo-epitope and also functioning as a TLR agonist) in addition to 100 micrograms each of αOX40 (also called anti-OX40) agonist antibody (functioning as an immune agonist and activating T cells) and αCTLA4 (also called anti-CTLA4) antibody (functioning as an immune checkpoint inhibitor and blocking immune suppressive responses) by three intratumoral injections (every other day) into the tumor. 100 μg of the TLR9 agonist CpG was used as a positive control treatment. Tumor growth of treated and untreated (distal) tumors was monitored for 28 days using digital calipers. (Figure 1A-1B) shows tumor volume (pc) up to day 14 post-challenge; (Figure 1C) shows Kaplan-Meier survival analysis up to day 28 post-challenge. Statistics, one-way ANOVA (tumor volume) or log-rank (KM); *** , p<0.001; **** p<0.0001.

[0078] Example 2 This example shows that isotype control antibodies are less effective than anti-CTLA4 and anti-OX40 in a bilateral tumor model. Female C57BL / 6 mice were implanted with B16F10 as in Example 1 for Figures 1A-1BH. Tumor treatments are indicated and included 100 μg of isotype control antibodies for anti-OX40 or anti-CTLA4 antibodies (rat anti-mouse IgG1k and Syrian hamster IgG, respectively) and yeast pressure homogenized ("pressure lysate, pL"). w303a, yeast null pL; 170, pTK170 pL; N803, human IL-15 superagonist, 1 μg. KM survival curves are shown for treated (Figure 2A) and untreated (Figure 2B) tumors. Statistics for Figure 2A: log-rank analysis of each group relative to PBS; *** p<0.001.

[0079] Example 3 This example shows that intratumoral injection of intact or lysed neoepitope yeast + αCTLA4 / αOX40 eradicates B16F10 tumors and confers tumor protection upon tumor rechallenge. Female C57BL / 6 mice were implanted with B16F10 cells and treated with triple therapy as described in Example 1 for Figures 1A-1H, except that the amount of antibody was lowered to 25 μg each and mixed with 5YU of intact (Int) or pressure homogenized (Lys) yeast. Figure 3A shows the tumor growth rate of the primary tumor, with complete response rates of 8 / 9 and 7 / 8 mice for intact and lysed yeast, respectively. Figure 3B shows that complete responders were rechallenged with 130,000 B16F10 tumor cells on day 43 and growth was monitored for an additional 94 days. An additional exploratory group of 5 naive mice was vaccinated subcutaneously with 2.5YU intact and 2.5YU pressure homogenized pTK170 yeast and 25μg of each antibody was delivered intraperitoneally at the same time (grey). Statistics by 2-way ANOVA with Dunnett's multiple comparisons: Figure 3A, p<0.0001, intact and lysate groups compared to PBS from day 8 onwards; Figure 3B, intact and lysate groups compared to naive from day 21 onwards.

[0080] Example 4 This example shows the effect of αOX40 and αCTLA4 antibody dose on anti-tumor activity in the B16F10 triple therapy model. Female C57BL / 6 mice were implanted with B16F10 cells and treated with triple therapy as described in Example 1 for Figures 1A-1H, except varying amounts of αOX40 and αCTLA4 antibodies were tested at 10-100 μg each as indicated. Antibodies were mixed with 5YU of pressure homogenized yeast (lysate). Primary tumor growth rates through days 8 (Figure 4A) and 17 (Figure 4B) post-treatment are shown. Statistics by two-way ANOVA with Dunnett's multiple comparisons compared to PBS; significance applies to all lysate cohorts. *** 、 p<0.001; **** , p<0.0001.

[0081] Example 5 This example shows the effect of αOX40 and αCTLA4 antibody dose on anti-tumor activity in the B16F10 triple therapy model. Female C57BL / 6 mice were implanted with B16F10 cells and treated with triple therapy as described in Example 1 for Figures 1A-1H, except varying αOX40 and αCTLA4 antibody doses were tested from 10 μg to 100 μg as indicated. Antibodies were mixed with 5YU of pressure homogenized yeast (lysate). Figure 5A shows the tumor growth rate (volume) up to day 52 after treatment; Figure 5B shows the Kaplan-Meier survival plot for the same period. As can be seen, very low doses of anti-Ox40 were required to obtain long-term protection. It is not necessary to have a systemic dose of anti-Ox40 to obtain activity.

[0082] Example 6 This example shows intratumoral injection of yeast presenting Fc-OX40L. Figures 6A-6B: Yeast was engineered to express surface-displayed protein G, in this case Fc-OX40L, which binds any Fc-containing protein. Figure 6A is a schematic of the strain; Figure 6B is flow cytometry analysis showing that after coating and washing of protein G-expressing yeast, approximately 54% of the yeast cells had detectable Fc-OX40L. Figure 6C shows the intratumoral injection regimen and treatment groups; assays were performed as in Example 1 for Figures 1A-1H, except that the specific concentrations of treatment components were varied as indicated. The effect of treatments on tumor growth and survival of treated (Figures 6D-6J) and untreated (Figures 6K-6Q) tumors is shown. For both Figures 6D-6I and 6K-6P, spider plots show tumor growth for individual mice, and Kaplan-Meier curves (Figures 6J and 6Q) show survival. Log-rank statistics, for comparisons shown ** p<0.01. EBY100, parent yeast strain; 424, empty yeast expression plasmid; Mox40L-Fc, mouse OX40L fused to IgG1 Fc.

[0083] Example 7 This example shows that N803 can replace αOX40 in a B16F10 intratumoral injection assay. Female C57BL / 6 mice were implanted intradermally with 130,000 B16F10 tumor cells in the right flank. When tumors were 7-8 mm in diameter, mice were treated three times (every other day) with αOX40 agonist antibody or N803, αCTLA4 antibody, and yeast null lysate. Tumor growth of injected (primary) tumors was monitored for 38 days (Figure 7A) or 68 days (Figure 7B).

[0084] Example 8 This example shows the reduction of MC38 tumor growth following αPDL1 / αOX40 / yeast intratumoral injection (it) therapy. Female C57BL / 6 mice were implanted intradermally with 300,000 MC38 tumor cells in the right flank. When tumors were approximately 3-4 mm in diameter, mice were treated three times (every other day) with 25 μg αOX40 agonist antibody and 25 or 100 μg αPD-L1 antibody + or - 5YU yeast null lysate. (Figures 8A-8E) Primary tumor growth was monitored for 42 days. On day 45, complete responders were re-challenged intradermally in the contralateral (left) flank with 300,000 MC38 tumor cells and tumor volume (Figures 8f-8J) and survival (Figure 8K) were monitored for an additional 48 days. W303a pL, pressure homogenized yeast null lysate.

[0085] Example 9 This example shows the titration of anti-PDL1 / anti-OX40 antibodies for intratumoral treatment of MC38 tumors. Female C57BL / 6 mice were implanted intradermally with 300,000 MC38 tumor cells in the right flank. When tumors were approximately 3-7 mm in diameter, mice were treated three times (every other day) with αOX40 agonist antibody and αPD-L1 antibody (range 0.25-25 μg) + 1 YU of yeast null lysate (Figure 9A). Survival was monitored for 48 days and showed a significant contribution of yeast lysate to the treatment effect (Figure 9B). W303a, yeast null; pL = pressure lysate. Statistics: Mantel-Cox test compared to PBS. * ;^Compared to a group with the same dose of antibody but lacking yeast pL.

[0086] Example 10 This example shows the strong therapeutic effect of aPDL1 / aOX40 + yeast lysate intratumoral therapy in a bilateral MC38 implantation model. Female C57BL / 6 mice were implanted intradermally with 300,000 MC38 tumor cells in the right flank (day 0) and left flank (day 2). On day 7, one tumor per mouse was treated three times (every other day) with 25 μg of αOX40 agonist antibody and αPD-L1 antibody + 5YU of yeast null lysate, respectively (Figure 10A). Growth impairment of treated (Figures 10B-10F) and untreated (Figures 10G-10K) tumors is shown, and cure rates are shown. (Figure 10L) Kaplan-Meier curve showing overall survival up to day 58 post-challenge (population data, treated and untreated tumors). Single tumors were 1000 mm 3 or when the combined treated and untreated tumor volume reaches 1500 mm 3 Mortality events (sacrifice) were recorded when the rats reached 0.01 mg / kg / day. Mantel-Cox statistics for treatment groups compared to PBS: ** , P < 0.01; **** , P<0.0001.

[0087] Example 11 This example illustrates the evaluation of anti-CTLA4, anti-PD-L1, N-803 and yeast (W303) in a two-tumor IT B16.F10 mouse model. Figure 11A shows a schematic of the IT injection regimen, and Figure 11B shows the treatment groups and the specific amounts of the various treatment components. The assay was generally performed as in Example 1. As shown in Figures 12A-12F, tumors injected with 50ug of anti-CTLA4 / anti-PD-L1 / N-803 / yeast were eliminated (see Figure 12A, 50ug injected tumor graph). As shown in Figures 13A-13F, IT injection of anti-CTLA4 / anti-PD-L1 / N-803 / yeast delayed tumor growth of uninjected distant tumors. Overall, the results in Figures 12A-12F and 13A-13F show that anti-CTLA4, anti-PD-L1, N-803 and yeast (W303) administered via IT injection result in a significant reduction in the growth of both injected and uninjected tumors.

[0088] Example 12 This example shows the efficacy comparison of intact yeast (INT) versus clarified lysate (CL) yeast (i.e., CL yeast is INT yeast that has been pressure lysed, centrifuged, and filtered) using a two-tumor B16.F10 mouse melanoma model as in Example 11. As seen in Figures 14A-14D, the INT yeast formulation is more effective at reducing tumor growth in both injected and uninjected tumors than the CL yeast formulation when combined with N-803 and a checkpoint inhibitor.

[0089] Example 13 This example shows the development of IT and IP / SC immunotherapy administration of anti-CTLA4 / anti-PD-L1 / N-803 / yeast in a two-tumor B16.F10 melanoma model as discussed in Example 11 (specifically, Groups 3 and 7 as described in Figure 11B). As shown in Figures 15A-15C, IT injection provides better tumor growth inhibition than the IP / SC administration route. Furthermore, Figure 16 shows that IT or IP / SC injection of anti-CTLA4 / anti-PD-L1 / N-803 / yeast does not affect mouse body weight.

[0090] Example 14 The objective of this example was to determine the most effective dosing schedule of N803 administered as an intratumoral injection with anti-CTLA4 and intact yeast (W303a). Figure 17 shows the 803 dose schedule in a combination IT therapy in a two-tumor B16.F10 melanoma model. As shown in Figures 18A-18D, co-injection of N-803 with anti-CTLA4 / yeast given three times every two days effectively inhibits tumor growth of injected tumors. Figure 19 shows a comparison of 2-day and 4-day N-803 dose intervals on the growth of injected tumors, with 2 days showing 8 / 9 cures, 4 days showing 6 / 8 cures, and 2 questionable. As shown in Figures 20A-20D, co-injection of N-803 with anti-CTLA4 / yeast given three times every two days effectively inhibits tumor growth of non-injected tumors. Figures 21A-21B show a comparison of 2-day and 4-day N-803 dose intervals on the growth of non-injected tumors.

[0091] Example 15 This example shows the evaluation of IT injected N-803 / anti-PD-L1 + yeast INT or yeast CL formulations in a two-tumor MC38 colorectal cancer model. Figure 22A shows a schematic of the IT injection regimen, and Figure 22B shows the specific amounts (in Vac) of the various treatment components along with the treatment groups and the equivalent YU amounts of yeast. As shown in Figures 23A-23E, yeast lysate (CL) in combination with anti-PDL1 / N-803 is the most effective yeast formulation for slowing the growth of injected tumors. As shown in Figures 24A-24A, INT yeast in combination with anti-PDL1 / N-803 is the most effective yeast formulation for slowing the growth of non-injected tumors.

[0092] While various embodiments of the present invention have been described in detail, it will be apparent that modifications and adaptations of these embodiments may occur to those skilled in the art, but it should be clearly understood that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

1. A composition for injection into a solid tumor, comprising an effective amount of yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

2. The composition of claim 1 , wherein the yeast is Saccharomyces cerevisiae.

3. The composition of claim 1 , wherein the yeast is genetically engineered to express a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

4. The composition of claim 1 , wherein the Ox40 or CD40 receptor agonist is a monoclonal antibody or a receptor ligand.

5. The composition of claim 1 , wherein the tumor is mucosal, melanoma, lung or colorectal.

6. The composition of claim 1 , wherein the tumor is metastatic.

7. The composition of claim 1 , wherein the yeast is genetically engineered to express Protein G on the surface of the yeast.

8. 8. The composition of claim 7, wherein the Ox40 or CD40 receptor agonist is an Fc-agonist fusion protein, and the Fc-agonist fusion protein is bound to the yeast prior to administration.

9. The composition of claim 1 , wherein the yeast is genetically engineered to express the immunostimulatory protein.

10. The composition of claim 1, wherein the IL-15 is nogapendekin-alfa-invaxcept (NAI; N-803).

11. The composition of claim 7, wherein the IL-15 is NAI.

12. 1. A method of treating a solid tumor, comprising intratumoral injection of an effective amount of a composition comprising yeast and / or a lysate thereof, a checkpoint inhibitor selected from the group consisting of an anti-CTLA-4 antibody and an anti-PDL1 antibody, and an immunostimulatory protein selected from the group consisting of an Ox40 receptor agonist, a CD40 receptor agonist, and an IL-15 superagonist.

13. 13. The method of claim 12, wherein the yeast is Saccharomyces cerevisiae.

14. The method of claim 12, wherein the yeast is genetically engineered to express a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

15. The method of claim 12, wherein the Ox40 or CD40 receptor agonist is a monoclonal antibody or a receptor ligand.

16. 13. The method of claim 12, wherein the tumor is mucosal, melanoma, lung or colorectal.

17. The method of claim 12, wherein the tumor is metastatic.

18. 13. The method of claim 12, wherein the yeast is genetically engineered to express Protein G on the surface of the yeast.

19. 19. The method of claim 18, wherein the Ox40 or CD40 receptor agonist is an Fc-agonist fusion protein, and the Fc-agonist fusion protein is bound to the yeast prior to administration.

20. The method of claim 12 , wherein the yeast is genetically engineered to express the immunostimulatory protein.

21. The method of claim 12, wherein the IL-15 is NAI.

22. The method of claim 18, wherein the IL-15 is NAI.

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