阅读说明：本技术 癌症治疗 (Cancer treatment ) 是由 格伦·马蒂 雅各布·坎品加 格雷厄姆·伯顿 安格斯·达格利什 于 2019-06-25 设计创作，主要内容包括：一种用于治疗、减少、抑制或控制检查点抑制剂难治性患者中一种或多种肿瘤的无活性全细胞分枝杆菌,其中所述检查点抑制剂难治性患者旨在与分枝杆菌的施用同时、分别或顺序进行检查点抑制治疗和/或共刺激检查点治疗,并且任选地进一步包括施用一种或多种其他抗癌治疗或药剂。(An inactive whole-cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy and/or co-stimulatory checkpoint therapy simultaneously, separately or sequentially to administration of the mycobacterium, and optionally further comprises administration of one or more other anti-cancer therapies or agents.)

1. An inactive whole cell Mycobacterium (Mycobacterium) for use in treating, reducing, inhibiting or controlling one or more tumors in a patient refractory to a checkpoint inhibitor, wherein the patient refractory to a checkpoint inhibitor is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the Mycobacterium.

2. The inactive whole-cell mycobacterium for use according to claim 1, wherein the checkpoint inhibition therapy comprises administration of one or more blocking agents selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single-chain variable fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof.

3. The inactive whole-cell mycobacterium for use according to claim 1 or 2, wherein the checkpoint inhibition therapy comprises administration of a sub-therapeutic amount and/or duration of the one or more blockers.

4. The inactive whole-cell mycobacterium according to any one of claims 1-3, wherein the one or more tumors are associated with sarcoma, preferably soft tissue sarcoma or non-soft tissue sarcoma.

5. The inactive whole-cell mycobacterium for use according to any one of claims 1-4, wherein the one or more tumors are associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors (carcinoma), head and neck cancer, and skin cancer.

6. The inactive whole-cell mycobacterium for use according to claim 5, wherein the one or more tumors are associated with pancreatic cancer, colorectal cancer, prostate cancer, skin cancer, or ovarian cancer.

7. The inactive whole-cell mycobacterium for use according to any one of claims 1-6, wherein the one or more tumors are metastatic.

8. The inactive whole-cell mycobacterium for use according to any one of claims 2-7, wherein the one or more blocking agents are selected from ipilimumab (ipilimumab), nivolumab (nivolumab), pembrolizumab (pembrolizumab), azetolizumab (ezolidumab), duvatuzumab (durvalumab), tremelimumab (tremelimumab), spatializumab (spatalizumab), avilamab (avelumab), sendilizumab (sintilimab), terlipab (torelizumab), MGA012, MGD013, MGD019, entilizumab (enolizumab), MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatllinmab (BMS 986), lagslab 525, IMP gn (simm 2810 (simm 281008, mei 289), pezilizumab (blet), pezilizumab), bevacizumab (blet) or a combination thereof, preferably pbc-55, pezilizumab (091), wherein the blocking agent (pezilizumab), MGA 3547, maculi, mexillizumab), the blocking agent (e) is selected from ipilimumab (PBF 986), pemphilizumab (e 3, pemphilizumab), preferably peglizumab), pemphilizumab (e 3, pemphilizumab), pemphilizumab (peglizumab), or a (e) or a (e) or a combination thereof, e, Nivolumitumumab.

9. The inactive whole-cell mycobacterium for use according to any one of claims 1-8, wherein the inactive whole-cell mycobacterium is a non-pathogenic heat-inactivated mycobacterium.

10. The inactive whole-cell mycobacterium for use according to any one of claims 1-9, wherein the inactive whole-cell mycobacterium is selected from the group consisting of: mycobacterium vaccae (M.vaccae), Mycobacterium obuense (M.obuense), Mycobacterium parafortuitum (M.parafortuitum), Mycobacterium aurum (M.aurum), M.indicus prandii, and combinations thereof.

11. The inactive whole-cell mycobacterium for use according to claim 10, wherein the inactive whole-cell mycobacterium is preferably a raw variant.

12. The inactive whole-cell mycobacterium for use according to any preceding claim, wherein the inactive whole-cell mycobacterium is for administration by parenteral, oral, sublingual, nasal or pulmonary route.

13. The inactive whole-cell mycobacterium for use according to claim 12, wherein the parenteral route is selected from subcutaneous (subdutaneous), intradermal, subdermal (subdermal), intraperitoneal or intravenous, preferably intradermal.

14. The inactive whole-cell mycobacterium for use according to claim 13, wherein the parenteral route comprises intratumoral, peritumoral, perilesional, or intralesional administration.

15. The inactive whole-cell mycobacterium for use according to any preceding claim, further comprising a co-stimulatory checkpoint treatment performed simultaneously, separately or sequentially with the administration of the mycobacterium, wherein the co-stimulatory checkpoint treatment comprises the administration of one or more binding agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scfvs), disulfide stabilized variable region fragments (dsfvs) or other antigen binding fragments thereof directed to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS and combinations thereof.

16. The inactive whole-cell mycobacterium for use according to claim 14, wherein the one or more binding agent is selected from the group consisting of utomiclumab, urelumab, MOXR0916, PF04518600, MEDI0562, GSK3174988, MEDI6469, RO7009789, CP870893, BMS986156, GWN323, JTX-2011, varilumab, MK-4166, NKT-214, and combinations thereof.

17. The inactive whole-cell mycobacterium for use according to any preceding claim, wherein the inactive whole-cell mycobacterium is administered before and/or after checkpoint inhibition therapy and/or co-stimulation checkpoint therapy.

18. The inactive whole-cell mycobacterium for use according to any preceding claim, further comprising the simultaneous, separate or sequential administration of one or more other anti-cancer treatments or agents with the mycobacterium administration and/or checkpoint inhibition therapy and/or co-stimulation checkpoint therapy.

19. The inactive whole-cell mycobacterium for use according to claim 18, wherein the one or more other anti-cancer treatments or agents are selected from: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, such as metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, such as OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, such as GVAX or CIMAvax.

20. The inactive whole-cell mycobacterium for use according to claim 19, wherein the TLR agonist comprises mifamurtide (mifamurtide) (Mepact), krestin (psk), MRx0518(4D Pharma), IMO-2125(tilsotolimod), CMP-001, MGN-1703 (lefetolimod), enterolimod, SD-101, GS-9620, imiquimod (imiquimod), resiquimod (resiquimod), MEDI4736, poly I: C, CPG7909, DSP-0509, VTX-2337(motolimod), MEDI9197, NKTR-262, G100, or PF-3512676, and combinations thereof, and wherein the chemotherapy comprises administration of one or more agents selected from: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mechlorethamine (mustine), vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, leucovorin (leucovorin), folinic acid, carboplatin, oxaliplatin, gemcitabine, folfororox, paclitaxel, pemetrexed, irinotecan, and combinations thereof.

21. The inactive whole-cell mycobacterium for use according to claim 19, wherein the chemotherapy comprises administration of one or more agents selected from the group consisting of: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mechlorethamine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, leucovorin, carboplatin, oxaliplatin, gemcitabine, folfinirox, paclitaxel, pemetrexed, irinotecan, and combinations thereof.

22. The inactive whole-cell mycobacterium of any one of claims 18-21, wherein the one or more other anti-cancer treatments or agents are administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intranasally, intrapulmonary, intrathecally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, stereotactically, orally, or by direct injection or infusion.

23. The inactive whole-cell mycobacterium for use according to any preceding claim, wherein the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance to checkpoint inhibitor treatment or acquired (secondary) resistance to checkpoint inhibitor treatment.

24. The inactive whole-cell mycobacterium for use according to claim 23, wherein the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance to treatment with the checkpoint inhibitor as a lack of response or an inadequate response to treatment with the checkpoint inhibitor for at least about 8 weeks or 12 weeks from the first dose.

25. The inactive whole-cell mycobacterium for use according to claim 23, wherein the checkpoint inhibitor-refractory patient exhibits acquired (secondary) resistance to treatment with the checkpoint inhibitor as evidenced by an initial response to the checkpoint treatment but subsequent recurrence and progression of one or more tumors.

26. The inactive whole cell mycobacterium for use according to any preceding claim, wherein the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance or acquired (secondary) resistance to treatment with one or more CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, LAG-3 inhibitors.

27. The inactive whole-cell mycobacterium for use according to any preceding claim, wherein the immunomodulator is to be administered into the skin of the individual by a microneedle device comprising a plurality of microneedles.

28. A method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from: cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof, directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcCR 3, and combinations thereof; and (ii) an inactive whole cell mycobacterium, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors or inactive whole cell mycobacterium alone.

29. The method of claim 28, wherein the method comprises administering a sub-therapeutic amount of the one or more checkpoint inhibitors.

30. The method of claim 28, wherein the one or more checkpoint inhibitors are selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scfvs), disulfide stabilized variable region fragments (dsfvs), or other antigen binding fragments thereof, and combinations thereof, directed against CTLA-4, PD-1, or PD-L1.

31. The method of any one of claims 28-30, wherein the one or more blocking agents are selected from ipilimumab, nivolumitumumab, pembrolizumab, azetolizumab, covelliuzumab, tremelimumab, sibatuzumab, abadazumab, avilimumab, certralizumab, teriprizumab, MGA012, MGD013, MGD019, entilizumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatllinab (986016), LAG525, IMP321, REGN2810 (cimipilimumab), REGN3767, pexitansinib, 3022855, FPA a008, BLZ945, GDC0919, ipastatin, indoximid, BMS986205, CPI-444, MEDI 3747, PBF509, liriluzumab, and combinations thereof, preferably wherein the blocking agent is selected from one or more ipilimumab, preferably one or a combination thereof.

32. The method of any one of claims 28-31, wherein the checkpoint inhibition therapy further comprises a co-stimulatory checkpoint therapy performed simultaneously, separately or sequentially with administration of the mycobacterium, wherein the co-stimulation checkpoint treatment comprises administration of one or more binding agents selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof directed against CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS and combinations thereof, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors, co-stimulatory checkpoint therapy, or inactive whole cell mycobacteria alone.

33. The method of claim 32, wherein the one or more binding agent is selected from the group consisting of utomicumab, uremicab, MOXR0916, PF04518600, MEDI0562, GSK3174988, MEDI6469, RO7009789, CP870893, BMS986156, GWN323, JTX-2011, varliumab, MK-4166, NKT-214, and combinations thereof.

34. The method of any one of claims 28-33, further comprising administering one or more additional anti-cancer therapies or agents simultaneously, separately, or sequentially with the administration of the mycobacterium, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors, co-stimulatory checkpoint therapies, one or more additional anti-cancer therapies or drugs, or inactive whole cell mycobacteria alone.

35. The method of claim 34, wherein the one or more additional anti-cancer treatments or agents are selected from the group consisting of: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, such as metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, such as OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, such as GVAX or CIMAvax.

36. The method of claim 35, wherein the TLR agonist comprises melpact, krestin (psk), MRx0518(4D Pharma), IMO-2125(tilsotolimod), CMP-001, MGN-1703(lefitolimod), enterolimod, SD-101, GS-9620, imiquimod, resiquimod, MEDI4736, poly I: C, CPG7909, DSP-0509, VTX-2337(motolimod), MEDI9197, NKTR-262, G100, or PF-3512676, and combinations thereof.

37. The method of claim 35, wherein the chemotherapy comprises administration of one or more agents selected from the group consisting of: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mechlorethamine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, leucovorin, carboplatin, oxaliplatin, gemcitabine, folfinirox, paclitaxel, pemetrexed, irinotecan, and combinations thereof.

38. The method of claims 34-37, wherein the one or more additional anti-cancer treatments or agents are administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intranasally, intrapulmonary, intrathecally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, stereotactically, orally, or by direct injection or infusion.

39. The method of any one of claims 28-38, wherein the neoplasia, tumor or cancer is associated with a sarcoma, preferably a soft tissue sarcoma or a non-soft tissue sarcoma.

40. The method of any one of claims 28-39, wherein the neoplasia, tumor or cancer is associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, and skin cancer.

41. The method of claim 40, wherein the neoplasia, tumor or cancer is associated with pancreatic cancer, colorectal cancer, prostate cancer, skin cancer or ovarian cancer.

42. The method of claim 28 or any one of claims 40 or 41, wherein the neoplasia, tumor or cancer is metastatic.

43. The method of any one of claims 28-42, wherein the inactive whole cell Mycobacterium is selected from the group consisting of: mycobacterium vaccae, Mycobacterium obuense, Mycobacterium parafortuitum, Mycobacterium aurum, M.indicus pranii, Mycobacterium phlei (M.phlei), and combinations thereof.

44. The method of any one of claims 28-43, wherein the inactive whole cell Mycobacterium is a raw variant.

45. The method of any one of claims 28-44, wherein the inactive whole cell Mycobacterium and/or checkpoint inhibitor is administered by a parenteral, oral, sublingual, nasal or pulmonary route.

46. The method of claim 45, wherein the parenteral route is selected from subcutaneous, intradermal, subdermal, intraperitoneal, or intravenous.

47. The method of claim 45 or 46, wherein the parenteral route comprises intratumoral, peritumoral, perilesional, or intralesional administration.

48. The method of any one of claims 28-47, wherein the concentration is about 10⁴To about 10¹⁰A cell, preferably about 10⁷To about 10⁹The inactive whole-cell mycobacteria are administered in an individual cell amount.

49. The method of any one of claims 28-48, wherein the enhanced therapeutic effect is measured by increased overall survival time.

50. The method of any one of claims 28-49, wherein enhanced therapeutic effect is measured by increased progression-free survival.

51. The method of any one of claims 28-50, wherein the enhanced therapeutic effect is measured by reducing or stabilizing the tumor size of one or more of the tumors, as defined by RECIST1.1, comprising disease Stabilization (SD), Complete Remission (CR), or Partial Remission (PR) of a tumor of interest; and/or Stable Disease (SD) or Complete Remission (CR) of one or more non-target tumors.

52. The method of any one of claims 28-51, wherein enhanced therapeutic effect is measured by improved overall remission rate and/or improved quality of life.

53. The method of any one of claims 28-52, wherein the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance to checkpoint inhibitor treatment or acquired (secondary) resistance to checkpoint inhibitor treatment.

54. The method of claim 53, wherein the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance to checkpoint inhibitor treatment as evidenced by a lack of response or an inadequate response to the checkpoint inhibitor treatment lasting at least about 8 weeks or 12 weeks.

55. The method of claim 54, wherein the checkpoint inhibitor-refractory patient exhibits acquired (secondary) resistance to checkpoint inhibitor treatment as evidenced by an initial response to the checkpoint inhibitor treatment but a subsequent recurrence and progression of one or more tumors.

56. The method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in a checkpoint inhibitor refractory individual of any one of claims 28-55, wherein the method comprises:

(i) providing a microneedle device comprising a plurality of microneedles,

(ii) causing the microneedles to penetrate the skin of the individual and assume an anchored state in which the microneedles are anchored in the skin and extend from the microneedle device,

(iii) delivering an amount of an immunomodulatory agent into the skin through the microneedle, wherein the immunomodulatory agent comprises whole cell, inactive mycobacteria.

57. A kit of parts for delivering at least one immunomodulator into the skin of a checkpoint inhibitor refractory patient, comprising:

a microneedle device comprising a plurality of microneedles, and

one or more immune modulators selected from the group consisting of:

whole-cell inactive mycobacteria such as Mycobacterium vaccae such as NCTC 11569, Mycobacterium obuense such as NCTC 13365, Mycobacterium parafortuitum, Mycobacterium aurum, M.indicus pranii, Mycobacterium phlei, and combinations thereof, and;

a checkpoint inhibitor selected from a cell, protein, peptide, antibody or antigen-binding fragment thereof against CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7-H3, B7-H4, B7-H6, A2AR, or IDO, and combinations thereof.

Technical Field

The present invention relates to the field of cancer therapy. In particular, the present invention relates to methods of preventing, treating, or inhibiting the development of tumors and/or metastases in individuals refractory (resistance) checkpoint inhibitor therapy.

Background

In humans with advanced cancer, anti-tumor immunity is often ineffective due to the interplay of proinflammatory and anti-inflammatory, immunostimulatory, and strict regulation of immunosuppressive signals. It is now believed that the immune system constantly monitors and eliminates newly transformed cells. Thus, cancer cells change their phenotype to cope with immune pressure, thereby evading attack (immune editing) and up-regulating the expression of inhibitory signals. Primary tumors and metastases maintain their own survival through immunoediting and other destructive procedures. One of the major mechanisms of anti-tumor immune destruction is "T cell depletion", which is caused by long-term exposure to antigens and is characterized by inhibitory receptor upregulation. These inhibitory receptors act as checkpoints to prevent uncontrolled immune responses.

PD-1 and co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4(CTLA-4), B-lymphocyte and T-lymphocyte attenuating agents (BTLA; CD272), T-cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3; CD223), and the like, are often referred to as checkpoint regulators. They function as molecular "toll booths" allowing extracellular information to indicate whether cell cycle progression and other intracellular signaling processes should proceed.

In addition to specific antigen recognition by the TCR, T cell activation is also regulated by a balance between positive and negative signals emitted by co-stimulatory receptors. These surface proteins are typically members of the TNF receptor or B7 superfamily. Agonistic antibodies to activating costimulatory molecules and inhibitory antibodies to negative costimulatory molecules will enhance T cell stimulation, thereby inducing oncolysis.

Programmed cell death protein 1(PD-1 or CD279), a 55-kD type 1 transmembrane protein, is a member of the CD28 family of T cell co-stimulatory receptors, including immunoglobulin superfamily members CD28, CTLA-4, inducible costimulatory factor (ICOS) and BTLA. PD-1 is highly expressed on activated T cells and B cells. Different expression levels of PD-1 expression can also be detected on subsets of memory T cells. Two specific ligands for PD-1 have been identified: programmed death ligand 1(PD-L1, also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD 273). Both PD-L1 and PD-L2 have been shown to down-regulate T cell activation following binding to PD-1 in both mouse and human systems (Okazaki et al, Int Immunol, 2007; 19: 813-824). PD-1 interacts with its ligands PD-L1 and PD-L2 expressed on Antigen Presenting Cells (APCs) and Dendritic Cells (DCs), delivering a negative regulatory stimulus, thereby down-regulating the activated T cell immune response. Blocking PD-1 inhibits this negative signal and thus enhances the T cell response.

Numerous studies have shown that: the cancer microenvironment governs the PD-L1-/PD-1 signaling pathway; induction of PD-L1 expression is associated with suppression of anti-cancer immune responses and thus allows cancer progression and metastasis. For a variety of reasons, the PD-L1/PD-1 signaling pathway is the primary mechanism of immune escape from cancer. First and foremost, this pathway is involved in the down-regulation of the immune response of activated T effector cells present in the periphery. Second, PD-L1 is upregulated in the cancer microenvironment, while PD-1 is also upregulated on activated tumor-infiltrating T cells, which may therefore potentiate the vicious cycle of inhibition. Third, the pathway is complexly involved in innate immunity and adaptive immune regulation through bidirectional signaling. These factors make the PD-1/PD-L1 complex a central point through which cancer can manipulate immune responses and promote its own progression.

The first immune checkpoint inhibitor tested in clinical trials was ipilimumab (Yervoy, Bristol-Myers Squibb), a CTLA-4 mAb (mAb). anti-CTLA-4 mabs are powerful checkpoint inhibitors that eliminate "break" from naive and antigen-exposed cells. The treatment enhances the anti-tumor function of CD8+ T cells, increases the ratio of CD8+ T cells to Foxp3+ regulatory T cells, and inhibits the inhibitory function of the regulatory T cells. The major drawback of anti-CTLA-4 monoclonal antibody therapy is the development of autoimmune toxicity.

TIM-3 was identified as another important inhibitory receptor expressed by depleted CD8+ T cells. In a mouse cancer model, the most dysfunctional tumor-infiltrating CD8+ T cells have been shown to actually co-express PD-1 and T Μ -3.

LAG-3 is another recently identified inhibitory receptor that acts to limit effector T cell function and enhance the inhibitory activity of regulatory T cells. It was recently found that PD-1 and LAG-3 are widely co-expressed by tumor-infiltrating T cells in mice, and that the combined blockade of PD-1 and LAG-3 elicits a strong synergistic anti-tumor immune response in a mouse cancer model.

Antagonist mabs against PD-1 and its ligand PD-L1 are currently in different stages of development for cancer therapy, and recent human trials have shown that outcome is happy in cancer patients with advanced disease.

The first agent entering phase I clinical trials to block the B7-H1/PD-1 pathway was Nivolumab (Nivolumab) (MDX-1106/BMS-936558/ONO-4538), a fully human lgG4 anti-PD-1 monoclonal developed by Bristol-Myers Squibb.

Results of phase II studies showed that comparing the combination of nivolumab and ipilimumab with ipilimumab alone, objective remission rates for the combination treatment were 61%, objective remission rates for the monotherapy treatment were 11%, and complete remission rates were 22% and 0% respectively in patients with BRAF wild-type melanoma. Grade 3 or 4 treatment-related adverse events were reported in 54% of patients in the combination group and grade 3 or 4 treatment-related adverse events were reported in 24% of patients in the ipilimumab group. Any level of treatment-related adverse events that led to discontinuation of study drug occurred in 7.7% of patients in the nivolumab group, 36.4% of these patients in the nivolumab-ca-ipilimumab group, and 14.8% of these patients in the ipilimumab group.

Immune checkpoint inhibitor therapy is particularly successful in melanoma, and currently approved therapies for melanoma include anti-PD-1 (nivolumitumumab and pembrolizumab), anti-CTLA-4 (ipilimumab), and anti-PD-1/CTLA-4 combination regimens (nivolumitumumab-ipilimumab). Long-term survival data for melanoma patients treated with ipilimumab (anti-CTLA-4) indicates that 5-10 years after treatment initiation, there is evidence that 20% of patients show sustained persistent disease control or response. Remission rates at 3 years were 33% in melanoma patients treated with pembrolizumab (anti-PD-1), with 70% -80% of patients initially responding maintaining clinical efficacy.

A phase III study showed that median PFS was elevated in patients treated with nivolumab and ipilimumab (11.5 months; HR, 0.42, P <0.001) and nivolumab (6.9 months; HR, 0.57, P <0.001) alone compared to ipilimumab alone (2.9 months). Median OS of the combination group or the nivolumab group alone was not achieved in at least 28 months of follow-up, whereas ipilimumab alone was 20 months [ HR: combination vs. ipilimumab, 0.55(P < 0.0001); nivolumizumab vs. ipilimumab, 0.63(P < 0.0001); reference 16 ]. In the combination group, nivolumetrizumab group and ipilimumab group, the OS rates for two years were 64%, 59% and 45%, respectively.

The results of these clinical trials highlight the significant impact that immunotherapy has on the clinical management of patients with advanced metastatic melanoma. However, although approximately 35% to 60% of patients have RECIST (solid tumor efficacy evaluation criteria) efficacy (10% -12% complete remission) on anti-PD-1 based immunotherapy, it has been shown that 40% to 65% of patients initially have little or no RECIST efficacy, and 43% of responders develop acquired resistance within 3 years.

Through analysis of clinical trial data, three broad categories of patients can be identified-1 patients who initially respond and continue to respond (responders), (2) patients who never respond (congenital resistance), and (3) patients who initially respond but ultimately lead to disease progression (acquired resistance).

Thus, despite the unprecedented rate of persistent remission observed in cancer immunotherapy, most patients do not benefit from treatment (primary resistance), and some responders relapse after a period of response (acquired resistance). Several common cancer types show very low response frequency (breast, prostate and colon cancers), and even heterogeneous responses are found between different tumors within the same patient. The mechanisms of innate and acquired resistance to checkpoint inhibitor therapy are not fully understood, in part because of the incomplete understanding of the full complement of clinical, molecular and immunological factors associated with the clinical efficacy and long-term benefit of checkpoint inhibitor therapy. Furthermore, few pre-clinical models with robust immune function can induce tumor regression through checkpoint inhibitors, thereby limiting the reproducibility of tumor-immune interaction diversity in patients.

Patients with primary resistance to checkpoint inhibitors do not respond to the initial treatment. Ongoing research has shown that both tumor cell endogenous and tumor cell exogenous factors are involved in resistance mechanisms.

Factors that contribute to primary or adaptive resistance include: lack of antigenic mutations, T cell failure, lack of adequate or appropriate tumor antigen presentation and/or processing, impaired DC maturation, loss of HLA expression, alterations of several signaling pathways (MAPK, PI3K, JAK, STAT, WNT, IFN), IDO induction, CD73 upregulation, constitutive expression of PD-L1, impaired intratumoral immune cell infiltration (e.g., T cells), activation of other immune suppression checkpoints (e.g., VISTA, LAG, TIM-3), activation of metabolic/inflammatory mediators, overexpression of VEGF, and activation of immune suppression cells (e.g., tumor-associated macrophages (TAMs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), etc.).

Other factors associated with acquired cancer resistance include target antigens, loss of HLA and changes in interferon signaling, and loss of or epigenetic changes in T cell function.

Immunosuppressive cell types that have been shown to affect checkpoint inhibitor efficacy in preclinical models include Treg, MDSC, Th2 CD4^tT cells and M2 polarized tumor-associated macrophages. These cell types individually or collectively promote the immunosuppressive Tumor Microenvironment (TME), primarily by releasing cytokines, chemokines, and other soluble mediators to prevent antitumor cytotoxicity and Th 1-directed T cell activity. Experiments have shown that depletion of these immunosuppressive cell types (e.g., MDSCs and tregs) can enhance the anti-tumor immune response, overcoming innate resistance.

Higher tregs in tumor tissue: teffector cell (Teff) ratios are associated with poor prognosis in many cancers, including ovarian cancer, pancreatic ductal adenocarcinoma, lung cancer, glioblastoma, non-hodgkin's lymphoma, melanoma, and other malignancies. Thus, for tumors whose treatment fails to increase Teff and/or deplete tregs to increase the ratio of Teff to tregs, it is possible to be resistant to treatment either early in the treatment or during the disease relapse background (during the delayed disease setting).

It is therefore an object of the present invention to provide a combination therapy for the treatment of cancer in a patient identified as resistant to checkpoint inhibitor treatment. The combination therapy comprises inactive whole cell mycobacteria and blockade of checkpoint inhibitors, wherein the therapy has the potential to overcome this innate or acquired resistance to treatment with checkpoint inhibitors.

Disclosure of Invention

The present invention provides an effective method for treating and/or preventing the establishment of cancer and/or metastases in patients refractory to checkpoint inhibitors by administering a checkpoint inhibitor that acts synergistically with an inactive whole cell mycobacterium.

In a first aspect of the invention, there is provided an inactive whole cell Mycobacterium (Mycobacterium) for use in the treatment, reduction, inhibition or control of one or more tumours in a patient refractory to a checkpoint inhibitor, wherein the checkpoint inhibitor-refractory patient is intended to be treated for checkpoint inhibition simultaneously, separately or sequentially with administration of the Mycobacterium.

In a second aspect of the invention, there is provided an inactive whole-cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the checkpoint inhibition therapy comprises administration of one or more blockers selected from the group consisting of cells, proteins, peptides, antibodies, ADC (drug-antibody), Fab-fragments (Fab-conjugate), Fab-fragments (Fab-antibody), and combinations thereof directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof, F (ab')2 fragments, diabodies, triabodies, tetrabodies, probodies, single chain variable fragments (scFv), disulfide stabilized variable region fragments (dsFv) or other antigen binding fragments thereof.

In a third aspect of the invention, there is provided an inactive whole-cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, and further comprising co-stimulatory checkpoint therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the co-stimulatory checkpoint therapy comprises administration of one or more binding agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scFv), scFv fragments, and combinations thereof to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, and combinations thereof, Disulfide stabilized variable region fragments (dsFv) or other antigen binding fragments thereof.

In a fourth aspect of the invention, there is provided an inactive whole-cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, optionally further comprising co-stimulatory checkpoint therapy, and further comprising simultaneous, separate or sequentially with administration of the mycobacterium and/or checkpoint inhibition therapy and/or co-stimulatory checkpoint therapy administration of one or more additional anti-cancer therapies or agents, wherein the one or more additional anti-cancer therapies or agents are selected from: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, for example metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, for example OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR4, TLR7, TLR8 or TLR9 agonists, or TLR5 agonists such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, for example GVAX or CIMAvax.

In a fifth aspect of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof, directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof; and (ii) an inactive whole cell mycobacterium, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors or inactive whole cell mycobacteria alone, and optionally wherein the method comprises administration of a sub-therapeutic amount of the checkpoint inhibitor.

In a sixth aspect of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof, directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof; (ii) (ii) an inactive whole-cell mycobacterium, and (iii) a co-stimulatory checkpoint treatment, wherein the co-stimulatory checkpoint treatment comprises administration of one or more binding agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single-chain variable fragments (scfvs), disulfide-stabilized variable region fragments (dsfvs), or other antigen-binding fragments thereof, directed to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, and combinations thereof, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors, co-stimulatory checkpoint treatment, or inactive whole-cell mycobacterium alone.

In a seventh aspect of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof, directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof; (ii) (ii) inactive whole cell mycobacteria, and (iii) administering one or more other anti-cancer treatments or agents selected from: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, e.g. metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, e.g. OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR4, TLR7, TLR8 or TLR9 agonists, or TLR5 agonists such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, e.g. GVAX or CIMAvax, wherein relative to the administration of one or more checkpoint inhibitors alone, co-stimulatory therapy, one or more other anti-cancer therapies or drugs, or inactive whole cell mycobacteria, the method results in enhanced therapeutic effect.

Drawings

The invention is described with reference to the following drawings, in which:

figure 1 shows the effect of a heat-inactivated Mycobacterium obuense (NCTC 13365) (IMM-101) preparation with or without co-administration of a checkpoint inhibitor (anti-PD-L1 mab) in a pancreatic cancer xenograft model (subcutaneous KPC cells).

Figure 2 shows the effect of a heat-inactivated mycobacterium obuense (NCTC 13365) (IMM-101) preparation with or without co-administration of a checkpoint inhibitor (anti-PD-1 mab) in a syngeneic breast cancer mouse model (subcutaneous EMT-6 cells) as detailed in example 3, wherein the figure shows the mean tumor volume +/-SE over time.

Figure 3 shows the proportion of CD3+ CD8+ cells and FoxP3Treg cells infiltrating the B16F10 tumor in control mice, mice treated with anti-CTLA-4 alone or in mice treated with a combination of IMM-101 and anti-CTLA-4 as detailed in example 2. We found a significant increase in this group (Anova p < 0.05). The lower panel shows the proportion of tumor-infiltrating CD3+ CD8+ cells and FoxP3Treg cells in control mice, mice treated with anti-PD-1 alone or in mice treated with a combination of IMM-101 and anti-PD-1.

FIG. 4 shows a schematic of the study of the effect of anti-PD-1 or anti-PD-1 antibodies with IMM-101 in a mouse model of breast cancer using the EMT-6 cell line, as detailed in example 3.

Figure 5 shows the effect of vehicle, anti-PD-1 antibody or combination of anti-PD-1 antibody and IMM-101 on tumor volume changes in a mouse model of breast cancer using the EMT-6 cell line as detailed in example 3.

Figure 6 shows the effect on the CD8/Treg ratio following administration of vehicle, anti-PD-1 antibody or anti-PD-1 antibody with IMM-101 in a mouse model of breast cancer using the EMT-6 cell line as detailed in example 3.

FIG. 7 shows the effect on INF- γ/IL-10 ratio following administration of vehicle, anti-PD-1 antibody or anti-PD-1 antibody with IMM-101 in a mouse model of breast cancer using EMT-6 cell line as detailed in example 3.

FIG. 8 shows a schematic of a study using Wild Type (WT) and Batf-/-mice, and graphical data of the effect of CD103+ DC on INF- γ release in WT and Batf-/-mice following subcutaneous injection of IMM-101.

FIG. 9 shows the effect of subcutaneously administering a preparation of heat-inactivated Mycobacterium obuense (NCTC 13365) (IMM-101) in the vicinity of a tumor with or without co-administration of a checkpoint inhibitor (anti-PD-1 mab) in a mouse model of checkpoint resistant melanoma (B16F10), showing the effect on mean tumor volume +/-SE over time.

Fig. 10 is the same as fig. 9, but where the figure shows the effect on mean tumor volume without SE (error-free line).

FIG. 11 is the same as FIG. 9, but wherein the graph shows the effect on mean tumor volume +/-SE until day 16 of the study.

Fig. 12 is the same as fig. 9, but wherein the graph shows the effect on median tumor volume.

Figure 13 is the same as figure 9, but where the figure shows the effect on median tumor volume up to study day 16.

FIG. 14 is the same as FIG. 9, but wherein the graph is a Kaplan-Meier analysis survival plot.

FIG. 15 shows the effect of subcutaneously administering a preparation of heat-inactivated Mycobacterium obuense (NCTC 13365) (IMM-101) in the vicinity of a tumor with or without co-administration of a checkpoint inhibitor (anti-PD-1 mab) in a mouse model of checkpoint resistant pancreatic cancer (Pan02), where the graph shows the effect on mean tumor volume +/-SE over time.

Fig. 16 is the same as fig. 15, but where the figure shows the effect on mean tumor volume without SE (error-free line).

FIG. 17 is the same as FIG. 15, but wherein the graph shows the effect on mean tumor volume +/-SE until day 37 of the study.

Fig. 18 is the same as fig. 15, but wherein the graph shows the effect on median tumor volume.

Figure 19 is the same as figure 15, but wherein the figure shows the effect on median tumor volume up to study day 37.

Detailed Description

The present invention provides a method of treating, reducing, inhibiting or managing neoplasia, tumor or cancer in an individual refractory to checkpoint comprising administering an inactive whole cell mycobacterium and one or more checkpoint inhibitors. Based on the surprising discovery that administration of whole cell heat-inactivated mycobacteria in combination with an anti-PD-L1 antibody (checkpoint inhibitor) in an animal model of checkpoint inhibitor resistance can result in synergistic anti-tumor activity and/or more potent anti-tumor activity than either mycobacteria or anti-PD-L1 antibody alone. Furthermore, mycobacterial treatment has been shown to improve the activity of cytotoxic T lymphocytes in a number of animal models with different cancer cell lines. It is believed that this improved activity contributes to a reduction in innate and/or primary or adaptive resistance to treatment with checkpoint inhibitors.

Thus, while it is known in the art that some combination therapies may reverse or reduce innate and/or primary or adaptive resistance, the invention disclosed herein provides combination therapies that are optimized to improve therapeutic efficacy and thus response in a greater proportion of checkpoint refractory patients.

In order that the invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

A "checkpoint inhibitor" is an agent that acts on a surface protein that is a member of the TNF receptor or B7 superfamily, including an agent that binds to a negative costimulatory (co-inhibitory) molecule selected from CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcCR 3, and/or their respective ligands, including PD-L1. "blockers" are agents that bind to the above-mentioned negative co-stimulatory molecules and/or their respective ligands. "checkpoint inhibitor" and "blocker" are used interchangeably throughout.

The inactive whole-cell mycobacterium as defined in the present invention is a component that stimulates innate immunity and type 1 immunity, including Th1 and macrophage activation/polarization and cytotoxic cell activity, as well as independently downregulating inappropriate anti-Th 2 responses through immune regulatory mechanisms.

The terms "tumor," "cancer," and "neoplasia (neoplasma)" are used interchangeably to refer to a cell or population of cells whose growth, proliferation or survival is greater than that of a normal counterpart, e.g., a cell proliferative or differentiative disorder. Usually, growth is not controlled. The term "malignant" refers to affecting nearby tissue. The term "metastasis" refers to the spread or spread of a tumor, cancer, or neoplasia to other sites, locations, or regions within an individual, where the sites, locations, or regions are distinct from the primary tumor or cancer.

The terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1" and "PD 1" are used interchangeably and include variants, isoforms, species homologs of human PD-1 and analogs having at least one common epitope with PD-1.

The terms "OX 40", "CD 137", and "OX-40" are used interchangeably and include variants, isoforms, species homologs, and analogs of human OX40 that share at least one common epitope with OX 40.

The terms "GITR" and "glucocorticoid-induced TNFR family-related genes" are used interchangeably and include variants, isoforms, species homologs, and analogs of human GITR that share at least one epitope with GITR.

The terms "CD 137" and "4-1 BB" are used interchangeably and include variants, isoforms, species homologs, and analogs of human CD137 that share at least one common epitope with CD 137.

The terms "B7-H3" and "CD 276" are used interchangeably and include variants, isoforms, species homologs of human B7-H3 and analogs having at least one common epitope with B7-H3.

The terms "B7-H4" and "VTCN 1" are used interchangeably and include variants, isoforms, species homologs of human B7-H4 and analogs having at least one common epitope with B7-H4.

The terms "A2 AR" and "adenosine A2A receptor" are used interchangeably and include variants, isoforms, species homologs of human A2AR and analogs having at least one common epitope with A2 AR.

The terms "IDO" and "indoleamine 2, 3-dioxygenase" are used interchangeably and include variants, isoforms, species homologs, and analogs of human IDO having at least one common epitope with IDO.

The terms "cytotoxic T lymphocyte-associated antigen-4", "CTLA 4" and "CTLA-4 antigen" are used interchangeably and include variants, isoforms, species homologs of human CTLA-4 and analogs having at least one common epitope with CTLA-4.

As used herein, "subtherapeutic amount" refers to a dose or treatment duration of a therapeutic compound (e.g., an antibody) that is lower than the conventional or typical dose or treatment duration of the therapeutic compound when administered alone for the treatment of cancer. Typical dosages of known therapeutic compounds are known to those skilled in the art or can be determined by routine experimental work.

The term "therapeutically effective amount" is defined as the amount of checkpoint inhibitor in combination with inactive whole cell mycobacteria, which preferably results in a reduction of the severity of the symptoms of a cancer disease, an increase in the frequency and duration of the symptomatic phase of a cancer-free disease, or prevention of damage or disability caused by a affliction with the disease. The term "effective amount" or "pharmaceutically effective amount" refers to an amount of an agent sufficient to provide the desired biological or therapeutic result. The result may be a reduction, amelioration, remission, palliation, delay and/or remission of one or more of the signs, symptoms or causes of cancer, or any other desired alteration of a biological system. In the case of cancer, an effective amount may comprise an amount sufficient to cause tumor shrinkage and/or to reduce the rate of tumor growth (e.g., inhibit tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the development of, or prolong survival of, or induce stabilization of, a cancer or tumor. Preferably, the therapeutic effect is measured by reduction or stabilization of the tumor size of one or more of said tumors, as defined by RECIST1.1, including Stable Disease (SD), Complete Remission (CR) or Partial Remission (PR) of the tumor of interest; and/or Stable Disease (SD) or Complete Remission (CR) of one or more non-target tumors.

In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay relapse. A therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount of the drug or combination may bring about one or more of the following: (i) reducing the number of cancer cells; (ii) reducing the size of the tumor; (iii) inhibit, delay, slow, and preferably prevent, to some extent, cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of a tumor; and/or (vii) alleviate one or more symptoms associated with cancer to some extent. For example, for the treatment of a tumor, a "therapeutically effective amount" may induce a tumor shrinkage of at least about 5%, e.g., at least about 10%, or about 20%, or about 60% or more, relative to a baseline measurement. The baseline measurement may be from an untreated individual.

A therapeutically effective amount of a therapeutic compound can reduce tumor size, or reduce symptoms, in an individual. One of ordinary skill in the art will be able to determine such amounts based on factors such as the physical size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration selected.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement), which results in the selective damage, destruction, or clearance of cancer cells from the human body.

The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof.

The term "antigen-binding portion" of an antibody (or simply "antibody portion") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to a receptor and its ligand (e.g., PD-1), which include: (i) fab fragments, (ii) F (ab')2 fragments, (iii) Fd fragments consisting of VH and CHI domains, (iv) Fv fragments, (v) dAb fragments consisting of VH domains, and (vi) isolated Complementarity Determining Regions (CDRs). Single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and practical screening of the fragments is performed in the same manner as for intact antibodies.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

In addition to antibodies, other biomolecules may be used as checkpoint inhibitors, including peptides with binding affinity for the appropriate target.

The term "treatment" or "treating" refers to administering an active agent with the purpose of curing, healing, alleviating, relieving, altering, remedying, mitigating, ameliorating, or affecting a condition (e.g., disease), a symptom of a condition, or preventing or delaying the onset of a symptom, complication, biochemical symptom of a disease, or preventing or inhibiting further development of a disease, condition, or disorder in a statistically significant manner.

The term "individual" as used herein is intended to include both human and non-human animals. Preferred individuals include human patients in need of an enhanced immune response. The methods are particularly useful for treating human patients having disorders that can be treated by enhancing a T cell-mediated immune response. In a specific embodiment, the method is particularly suitable for treating cancer cells in vivo.

As used herein, the term "checkpoint inhibitor-refractory patient" refers to a patient identified as non-responsive to checkpoint inhibitor treatment. Refractory patients may exhibit congenital (primary) resistance to treatment with checkpoint inhibitors. No or insufficient response to treatment with the checkpoint inhibitor may demonstrate innate resistance for at least about 8 or 12 weeks from the first dose. Refractory patients may exhibit acquired (secondary) resistance to checkpoint inhibitor treatment. Acquired resistance can be evidenced by an initial response to the checkpoint treatment but subsequent recurrence and progression of one or more tumors. Checkpoint inhibitor refractory patients may be unresponsive to any checkpoint inhibitor, non-limiting examples include CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, LAG-3 inhibitors, or combinations thereof.

As used herein, the term "concurrently administering" or "concurrently" or "simultaneously" means that the administration is performed on the same day. The terms "sequential administration" or "order" or "separately" mean that the administration is performed on different days.

As defined herein, "simultaneous" administration includes administration of inactive whole cell mycobacteria and agents or procedures that include checkpoint inhibitor therapy and/or co-stimulatory checkpoint therapy and/or one or more other anti-cancer therapies or agents with an interval of about 2 hours or less than 1 hour from each other. Preferably, "simultaneous" administration refers to an agent or procedure in which an inactive whole cell mycobacterium and a checkpoint inhibitor therapy and/or a co-stimulatory checkpoint therapy and/or one or more other anti-cancer therapies or agents are administered simultaneously.

As defined herein, "administering" includes administering an agent or procedure comprising an inactive whole cell mycobacterium and a checkpoint inhibitor therapy and/or a costimulatory checkpoint therapy and/or one or more other anti-cancer therapies or agents more than about 12 hours, or about 8 hours, or about 6 hours or about 4 hours or about 2 hours apart.

As defined herein, "sequential" administration includes administration of inactive whole cell mycobacteria and an agent or procedure that includes a checkpoint inhibitor therapy and/or a co-stimulatory checkpoint therapy and/or one or more other anti-cancer therapies or agents, each multiple and/or multiple doses and/or on a non-identical occasion. The inactive whole cell mycobacterium may be administered to the patient before and/or after administration of the checkpoint inhibitor and/or co-stimulatory checkpoint therapy and/or one or more other anti-cancer therapies or agents. Alternatively, administration of inactive whole cell mycobacteria continues after checkpoint inhibitor therapy and/or co-stimulation checkpoint therapy and/or one or more other anti-cancer therapies or agent therapies.

The use of alternatives (e.g., "or") should be understood to mean either, two, or any combination of these alternatives. The indefinite articles "a" or "an" as used herein are understood to mean "one or more" of any stated or listed component.

As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per operation in the art. Alternatively, "about" may mean a range of up to 20%. Where particular values are provided in the present application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for the particular value.

Mycobacterium vaccae (m.vaccae) and mycobacterium obuense have been shown to induce complex immune responses in hosts. Treatment with these preparations will stimulate innate and type 1 immunity, including Th1 and macrophage activation as well as cytotoxic cell activity. They also independently down-regulate inappropriate Th2 responses through immune regulatory mechanisms. In experiments with mouse and human immune cells, it has been shown that IMM-101 (inactive whole cell Mycobacterium obuense) is a potent activator of antigen-presenting macrophages and Dendritic Cells (DCs), and that DC activation results in a typical type 1 immune response with increased production of the cytokine interferon- γ (IFN- γ) and the formation and activation of CD4+ T-helper 1 lymphocytes (Th1) and CD8+ Cytotoxic T Lymphocytes (CTLs) in lymph nodes where IMM-101 activated DCs are present.

In addition, other experiments have shown that IMM-101 also increases the number and activation of Natural Killer (NK) cells and T cells expressing gamma/delta receptors (gamma delta-T cells). Th1 and CTLs require tumor cells to express specific Tumor Associated Antigens (TAAs) for attack, whereas NK cells and γ δ -T cells do not require such TAAs to kill tumor cells. These four different immune cells work in concert to develop an effective anti-tumor response. With respect to cancer, the formation of IFN- γ -producing CTLs is likely the most important outcome of IMM-101 therapy, as the anti-tumor effects of IMM-101 observed in pancreatic cancer models can be completely abolished by depletion of CD8+ T cells.

The ability of IMM-101 to activate macrophages may not only assist DC activation by releasing proinflammatory macrophage-derived cytokines such as IL-12 required to turn DC off (skew) to a type 1 immune response, but may also be important in changing tumor-associated immunosuppressive type 2 macrophages to aggressive tumor type 1 macrophages. The latter feature is shown for a similar heat-inactivated mycobacterium m.

An important feature of IMM-101 is its ability to activate and mature DCs into a dendritic cell subset, i.e., cDC1 (i.e., the DCs required for a type 1 immune response). It has been shown that activating a sufficient number of cdcs 1 is a prerequisite for CPI to take effect.

It is widely believed that the type 1 immune response leading to the production of Th1 and CTL of INF- γ specifically attacking TAA expressing tumor cells, and activated NK cells and γ δ -T cells attacking tumors by other mechanisms, is a major mechanism and prerequisite for an effective anti-cancer response in humans, and thus should be the core of any immune-mediated cancer therapy-preclinical data suggesting that IMM-101 is capable of stimulating such desirable type 1 immune responses.

The effect of IMM-101 on DC priming has been studied in vitro, and IMM-101 was found to exhibit a dose-dependent capacity to induce phenotypic activation and cytokine production in human and murine DCs. For example, GM-CSF derived murine DCs exhibit dose-dependence on IMM-101, elevated membrane expression of CD80, CD86, CD40, and MHC II, while increased production of IL-6, IL-12p40, and nitric oxide, all of which are molecules essential for effective antigen-dependent activation of T cells. Furthermore, human monocyte-derived DCs showed a similar response to IMM-101, with CD80, CD86, and MHC II upregulated and secreted multiple relevant cytokines, indicating that activation of DCs was significant. In vitro exposure to IMM-101 also suggests that IMM-101 functionally affects DCs by enhancing their ability to process and present antigen.

In vivo experiments showed that 7 days after subcutaneous adoptive transfer of IMM-101 (in vitro) activated GM-CSF derived murine DCs to naive recipient mice, IMM-101 activated DCs were able to activate CD8+ and CD4+ T cells and promote IFN-. gamma.secretion by re-stimulating draining lymph node cell preparations.

Disclosed herein, the combination of several different checkpoint inhibitors (CPI) with inactive whole cell mycobacteria (IMM-101) has been studied in animal models using three different cancer cell lines (breast cancer, pancreatic cancer and checkpoint resistant melanoma B16F 10). In these experiments, CPI (or IMM-101) only treatment was only moderately to poorly effective in controlling primary tumor growth, but CPI in combination with IMM-101 showed a positive trend in breast cancer and was very effective in reducing tumor volume in pancreatic cancer. An increased CD8+/Treg ratio was found in breast tumors and melanomas. An increased IFN-. gamma./IL-10 (IL-10 is a cytokine produced by Tregs) ratio was also found in the spleen of the breast cancer model. These increased ratios are highly suggestive of improved CTL activity and decreased immunosuppressive activity, which is expected to result in a reduction or inhibition of primary and/or secondary resistance to checkpoint inhibitor treatment in human or animal individuals.

In one aspect of the invention, the inactive whole-cell mycobacteria includes whole-cell, non-pathogenic heat-inactivated mycobacteria. Examples of mycobacterium species for use in the present invention include mycobacterium bovis (m.vaccae), heat-resistant mycobacterium (m.thermoresistivile), mycobacterium microflavus (m.flavescens), mycobacterium durum (m.duvaliii), mycobacterium phlei (m.phereti), mycobacterium obuense (m.obuense), mycobacterium parafortis (m.parafortum), mycobacterium sphaericus (m.sphagni), mycobacterium loenium (m.aiciense), mycobacterium rhodesiae (m.rhodesiae), mycobacterium neogold (m.neoaureum), mycobacterium canescens (m.chuuense), mycobacterium eastern sea (m.tokaiiense), mycobacterium comosus (m.komosense), mycobacterium aureofaciens (m.aureouruum), M.w, mycobacterium tuberculosis (m.tucheriscus), mycobacterium africanum (m), mycobacterium africanum (m.gastrorhizophilus), mycobacterium tuberculosis (m.gastrogram-m), mycobacterium phleum (m.gastrogram-manicus), mycobacterium phlei (m) Minor mycobacteria (m.triviale), gordonia (m.gordonae), scrofula (m.scrofulaceae), mycobacterium paraffinum (m.paraffinicum), mycobacterium intracellulare (m.intercellulare), mycobacterium avium (m.avium), mycobacterium bufonis (m.xenopi), mycobacterium ulcerosa (m.ulcerans), mycobacterium dius (m.diernhoferi), mycobacterium smegmatis (m.smegmatis), mycobacterium Serpentis (m.thamnophilos), mycobacterium microflavus, mycobacterium fortuitum (m.fortuitum), mycobacterium exotica (m.peregrinum), mycobacterium (m.chelonei), mycobacterium paratuberculosis (m.paratuberculosis), mycobacterium leprae (m.leprae), and combinations thereof.

The inactive whole-cell mycobacteria are preferably selected from: mycobacterium vaccae, Mycobacterium obuense, Mycobacterium paratuberculosis, Mycobacterium aurum, M.indicus pranii, Mycobacterium phlei, and combinations thereof. More preferably, the inactive whole cell mycobacterium is a unprocessed (rough) variant.

The amount of mycobacterium administered to the patient is sufficient to elicit a protective immune response in the patient such that the patient's immune system is capable of generating an effective immune response against the cancer or tumor. In certain embodiments of the invention, a containment device is provided comprising an effective amount of a mycobacterium for use in the invention, which may be generally 10³To 10¹¹Organisms, preferably 10⁴To 10¹⁰Individual organism, more preferably 10⁶To 10¹⁰Individual organisms, even more preferably 10⁶To 10⁹An organism. The amount of mycobacteria used in the present invention is most preferably 10⁷To 10⁹Individual cells or organisms. Generally, for human and animal use, the composition according to the invention may be in the range of 10⁸To 10⁹Individual cell doses were administered. Alternatively, the dose is from 0.01mg to 5mg or from 0.01mg to 5mg of the organism, preferably from 0.1mg to 2mg or from 0.1mg to 2mg of the organism, more preferably the dose is about 1mg or 1mg of the organism. The dosage can be made into suspension or dry preparation.

M.indicus pranii, Mycobacterium vaccae and Mycobacterium obuense are particularly preferred.

The invention is useful for treating neoplastic diseases, such as solid or non-solid cancers. As used herein, "treating" includes preventing, reducing, managing and/or inhibiting neoplastic disease. These diseases include sarcomas, carcinoma (carcinoma), adenocarcinomas, melanomas, myelomas, blastomas, gliomas, lymphomas or leukemias. Exemplary cancers include, for example, malignant epithelial tumors, sarcomas, adenocarcinomas, melanomas, neuro (blastomas, gliomas), mesotheliomas, and reticuloendothelial, lymphoid, or hematopoietic neoplastic diseases (e.g., myelomas, lymphomas, or leukemias). In particular aspects, the neoplasia, tumor or cancer includes lung adenocarcinoma, lung cancer, diffuse or interstitial gastric cancer, colon adenocarcinoma, prostate adenocarcinoma, esophageal cancer, breast cancer, pancreatic cancer, ovarian adenocarcinoma, adrenal adenocarcinoma, endometrial adenocarcinoma, or uterine adenocarcinoma.

Neoplasias, tumors and cancers include benign, malignant, metastatic and non-metastatic types, including neoplasias, tumors or cancers at any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.), or in progression, worsening, stabilization or remission. Cancers that may be treated according to the present invention include, but are not limited to: bladder cancer, hematologic cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. In addition, the cancer may be specifically of the following histological type, although not limited to the following types: tumors, malignancies, undifferentiated carcinomas, giant and spindle cell carcinomas, small cell carcinomas, papillary carcinomas, squamous cell carcinomas, lymphoepithelial carcinomas, basal cell carcinomas, choriocarcinoma (pilomatrix carcinosa), transitional cell carcinomas, papillary transitional cell carcinomas, adenocarcinomas, gastrinomas, malignant cholangiocarcinomas, hepatocellular carcinomas, combined hepatocellular carcinomas and cholangiocarcinomas, trabecular adenocarcinomas, adenoid cystic carcinomas, adenocarcinomas in adenomatopolyps, adenocarcinomas, familial polyposis, solid carcinomas, carcinoids, malignant bronchioloalveolar adenocarcinomas, papillary adenocarcinomas, chromophobe carcinomas, eosinophil carcinomas, oxyphil adenocarcinomas, clear cell adenocarcinomas, granulomatous adenocarcinomas, follicular adenocarcinomas, papillary and follicular adenocarcinomas, non-sclerosing carcinomas, adrenocortical carcinomas, Skin adnociceptive adenocarcinoma, apocrine adenocarcinomas, sebaceous adenocarcinomas, ceruminous adenocarcinomas, mucoepidermoid carcinomas, cystadenocarcinomas, papillary serous cystadenocarcinomas, mucinous adenocarcinomas, signet 'cell carcinomas, invasive ductal carcinomas, medullary carcinomas, lobular carcinomas, inflammatory carcinomas, paget's disease, mammary glands, acinar cell carcinomas, adenosquamous carcinomas with squarosus, thymomas, malignant ovarian stromal tumors, malignant follicular carcinomas, malignant granulomatosis, malignant male cell tumors, malignant supportive cell carcinomas, testicular stromal tumors, malignant lipocytomas, malignant paragangliomas, malignant external paragangliomas of mammary glands (malignant extramalignant paramelanoma-malignant paramelanoma), malignant chromocytomas, hemangiomas, melanoma, and melanoma, Metastatic melanoma, malignant lentigo melanoma, nodular melanoma, acral lentigo melanoma, connective tissue proliferative melanoma, epithelioid melanoma, blue nevi, malignant sarcoma, fibrosarcoma, malignant mucinous sarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, muller's canal mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, interstitial tumor, malignant brenne tumor (malignant brentumur), malignant phyllodes tumor, malignant synovial sarcoma, mesothelioma, malignant clonal cell tumor, embryonal carcinoma, teratoma, malignant ovarian thyroid tumor, malignant membranous carcinoma, mesonephroma, malignant vascular sarcoma, vascular endothelial cell tumor, malignant Kaposi's sarcoma, malignant melanoma, malignant neuter's tumor, malignant neuter's cell tumor, malignant neuter's tumor, malignant melanoma, Hemangiopericyte tumor, malignant lymphangiosarcoma, osteosarcoma, paracortical osteogenic sarcoma, chondrosarcoma, chondroblastoma, malignant mesenchymal chondrosarcoma, giant cell tumor of bone, ewing's sarcoma, odontogenic fibroma, malignant enamel cell dental sarcoma, ameloblastic tumor, malignant enamel cell fibrosarcoma, pineal tumor, malignant chordoma, glioma, malignant ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, glioblastoma, oligodendroglioma, primitive neuroectodermal, cerebellar sarcoma, ganglionic neuroblastoma, retinoblastoma, olfactory neurogenic tumor, meningioma, malignant neurofibrosarcoma, schwannoma, malignant granulomatosis, malignant lymphoma, hodgkin's lymphoma, giant cell lymphoma, giant, Hodgkin's parasymoma, malignant lymphoma, small lymphocytic malignant lymphoma, large cell diffuse lymphoma, malignant lymphoma, follicular mycosis fungoides, other non-hodgkin's lymphoma as specified, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small bowel disease, leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic myeloid leukemia, eosinophilic myeloid leukemia, monocytic leukemia, mast cell leukemia, megakaryocytic leukemia, myeloid sarcoma, and hairy cell leukemia. Preferably, the cancer is selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer and soft tissue sarcoma and/or other forms of cancer. The tumor may be a metastatic or malignant tumor.

More preferably, the cancer is pancreatic cancer, colorectal cancer, prostate cancer, skin cancer, ovarian cancer or lung cancer.

In one embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended to be treated for checkpoint inhibition simultaneously, separately or sequentially with administration of the mycobacterium.

In one embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the checkpoint inhibition therapy comprises administration of one or more blockers selected from the group consisting of cells, proteins, peptides, antibodies, ADC (ADC-antibody) conjugates against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3 and combinations thereof, Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof.

In one embodiment of the invention, the checkpoint inhibition therapy comprises administration of a sub-therapeutic amount and/or duration of said one or more blockers.

In one embodiment of the invention, the one or more blocking agents are selected from ipilimumab (ipilimumab), nivolumab (nivolumab), pembrolizumab (pembrolizumab), azetolizumab (azetolizumab), duvatuzumab (durvalumab), tremelimumab (tremelimumab), spatializumab (sparotalizumab), avizumab (avelumab), siltilimab (sintilimab), terilizumab (torulalizab), MGA012, MGD013, MGD019, entilizumab (obenzitumab), MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, g453, relatllinab (mbbms 986016), mab525, IMP321, impdg 2810 (cimicim), pepilis 008, fprisc 2830251, betax 9855, piceid (blet), pbadix 986015, peglizumab (pegmati), pbd 989, pegmati ad 986015, and combinations thereof.

In one embodiment of the invention, the one or more blocking agents are preferably ipilimumab and/or nivolumab.

In one embodiment of the invention, checkpoint inhibitor treatment comprises administering in combination with inactive whole cell mycobacteria one or more blocking agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single-fragment (scFv), disulfide-stabilized variable region fragments (dsdisulfide bond) or other antigen binding fragments against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3 and combinations thereof, in order to reduce or inhibit primary tumor metastasis or other primary tumor metastasis to other cancers or other cancers, Or the formation or establishment of a metastatic tumor or cancer at a site remote from the primary tumor or cancer, thereby inhibiting or reducing the recurrence of the tumor or cancer or the progression of the tumor or cancer, preferably in a patient refractory to a checkpoint inhibitor.

In one embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the checkpoint inhibition therapy comprises administration of one or more blockers selected from the group consisting of cells, proteins, peptides, antibodies, ADC (ADC-antibody) conjugates against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3 and combinations thereof, Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv) or other antigen binding fragments, have the potential to elicit a potent and persistent immune response compared to any of the therapies alone, with enhanced therapeutic benefit, preferably as measured by reducing or stabilizing the tumor size of one or more of the tumors (as defined by RECIST 1.1), including Complete Remission (CR) or Partial Remission (PR) of the tumor of interest; and/or Stable Disease (SD) or Complete Remission (CR) of one or more non-target tumors.

In one embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in the manufacture of a medicament for the treatment of cancer in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium.

In another embodiment of the invention, there is provided a combination therapy for treating cancer in a patient refractory to a checkpoint inhibitor comprising an inactive whole cell mycobacterium which: (i) stimulating innate and type 1 immunity, including Th1 and macrophage activation and cytotoxic cell activity, and (ii) independently downregulating inappropriate Th2 responses through immune regulatory mechanisms; and a checkpoint inhibitor, optionally wherein the mycobacterium is selected from mycobacterium vaccae, mycobacterium obuense or m.indicus pranii.

In another embodiment of the invention, there is provided a combination therapy for treating cancer in a patient refractory to checkpoint inhibitors comprising inactive whole cell mycobacteria that mediate any combination of at least one of the following immunostimulatory effects on immunity: (i) increasing immune response, (ii) increasing T cell activation, (iii) increasing cytotoxic T cell activity, (iv) increasing NK cell activity, (v) increasing Th17 activity, (vi) alleviating T cell suppression, (vii) increasing pro-inflammatory cytokine secretion, (viii) increasing IL-2 secretion; (ix) increasing interferon-y produced by T cells, (x) increasing Th1 response, (xi) decreasing Th2 response, (xii) decreasing or eliminating at least one regulatory T cell (Treg), myeloid-derived suppressor cell (MDSC), iMC, mesenchymal stromal cells, monocytes expressing TIE2, (xiii) decreasing regulatory cell activity and/or the activity of one or more myeloid-derived suppressor cells (MDSC), iMC, mesenchymal stromal cells, monocytes expressing TIE2, (xiv) decreasing or eliminating M2 macrophages, (xv) eliminating pro-tumor activity of M2 macrophages, (xvi) decreasing or eliminating N2 neutrophils, (xvii) decreasing pro-tumor activity of N2 neutrophils, (xviii) decreasing inhibition of T cell activation, (xix) decreasing inhibition of CTL activation, (xx) decreasing inhibition of NK cell activation, (xxi) Reversing T cell depletion, (xxii) increasing T cell response, (xxiii) increasing activity of cytotoxic cells, (xxiv) stimulating an antigen-specific memory response, (xxv) inducing apoptosis or lysis of cancer cells, (xxvi) stimulating cytotoxicity or a resting effect on cancer cells, (xxvii) inducing direct killing of cancer cells, and/or (xxviii) inducing complement-dependent cytotoxicity and/or (xxix) inducing antibody-dependent cell-mediated cytotoxicity.

In another embodiment of the present invention, there is provided a combination therapy for treating cancer in a patient refractory to a checkpoint inhibitor, comprising an inactive whole cell mycobacterium that promotes CTL activity, wherein the CTL activity comprises secretion of one or more pro-inflammatory cytokines and/or CTL-mediated killing of target cells; and/or promote CD4+ T cell activation and/or CD4+ T cell proliferation and/or CD4+ T cell mediated cell depletion; and/or promote CD8+ T cell activation and/or CD8+ T cell proliferation and/or CD8+ T cell mediated cell depletion; and/or enhancing NK cell activity and/or NK cell proliferation and/or NK cell-mediated cell depletion, wherein enhanced NK cell activity comprises increased target cell depletion and/or pro-inflammatory cytokine release; and/or up-regulation or stimulation of CD103+ CD141+ DC; and/or reducing or eliminating the differentiation, proliferation, infiltration and/or activity of regulatory cells (tregs), and/or the differentiation, proliferation, infiltration and/or activity of myeloid-derived suppressor cells (MDSCs); and/or reducing or eliminating the target of inducible treg (itreg) infiltration.

In one embodiment of the invention, there is provided an inactive whole-cell mycobacterium for use in treating, reducing, inhibiting, or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy concurrently, separately, or sequentially with administration of mycobacterium, further comprising co-stimulatory checkpoint therapy concurrently, separately, or sequentially with administration of mycobacterium, wherein the co-stimulatory checkpoint therapy comprises administration of one or more binding agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scfvs), and combinations thereof to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, and combinations thereof, Disulfide stabilized variable region fragments (dsFvs) or other antigen binding fragments.

In one embodiment of the invention, the co-stimulation checkpoint treatment comprises administering one or more binding agents selected from the group consisting of utolimumab, urelimumab, MOXR0916, PF04518600, MEDI0562, GSK3174988, MEDI6469. rod7009789, CP870893, BMS986156, GWN323, JTX-2011, varlizumab, MK-4166, NKT-214, and combinations thereof.

In one embodiment of the invention, the inactive whole-cell mycobacterium is administered before and/or after checkpoint inhibition therapy and/or co-stimulation checkpoint therapy.

In another embodiment of the invention, there is provided an inactive whole-cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy concurrently, separately or sequentially with administration of the mycobacterium, further comprising administration of one or more additional anti-cancer therapies or agents concurrently, separately or sequentially with administration of the mycobacterium and/or checkpoint inhibition therapy and/or co-stimulatory checkpoint therapy.

In another embodiment of the invention, the one or more additional anti-cancer treatments or agents are selected from: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, for example metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, for example OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR3, TLR4, TLR7, TLR8 or TLR9 agonists, or TLR 051 5 agonists such as MRx 8(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, for example GVAX or CIMAvax.

In another embodiment of the invention, the one or more additional anti-cancer treatments result in an immunogenic cell death treatment, as described in WO 2013/07998. This treatment induces tumor immunogenic cell death, including apoptosis (type 1), autophagy (type 2), and necrosis (type 3), followed by release of tumor antigens, which are capable of inducing an immune response, including activation of cytotoxic CD8+ T cells and NK cells, and which may serve as targets, including making dendritic cells accessible to the antigens. Immunogenic cell death therapy can be performed at sub-optimal levels, i.e., non-curative therapy, such that it is not intended to completely remove or eradicate a tumor, but results in necrosis of certain tumor cells or tissues. The skilled person will appreciate that the degree of treatment required to achieve this depends on the technique used, the age of the patient, the condition of the disease and in particular the size and location of the tumour or metastasis. Particularly preferred treatments include: microwave radiation, targeted radiation therapy such as stereotactic ablation radiation (SABR), embolization (embolization), cryotherapy, ultrasound, high intensity focused ultrasound, radio-knife, hyperthermia, radiofrequency ablation, cryoablation, electrotome heating, hot water injection, alcohol injection, embolization (embolization), radiation irradiation, photodynamic therapy, laser beam irradiation, and combinations thereof.

In another embodiment of the invention, TLR agonists include MRx0518(4D Pharma), mifamurtide (Mepact), Krestin (PSK), IMO-2125(tilsotolimod), CMP-001, MGN-1703 (lefetolimod), entolimod, SD-101, GS-9620, imiquimod (imiquimod), resiquimod (resiquimod), MEDI4736, poly I: C, CPG7909, DSP-0509, VTX-2337(motolimod), MEDI9197, NKTR-262, G100, or PF-3512676, and combinations thereof.

In another embodiment of the invention, chemotherapy comprises administering one or more agents selected from the group consisting of: cyclophosphamide (cyclophosphamide), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), doxorubicin (doxorubicin), mechlorethamine (mustine), vincristine (vincristine), procarbazine (procarbazine), prednisolone (prednisolone), bleomycin (bleomycin), vinblastine (vinblastine), dacarbazine (carbazine), etoposide (etoposide), cisplatin (cispin), epirubicin (epirubicin), capecitabine (capecitabine), leucovorin (leucovorin), folinic acid (folinic acid), carboplatin (carboplatin), oxaliplatin (oxaliplatin), gemcitabine (gemcitabine), folfirx, paclitaxel (paclitaxel), medecine (irinotecan), irinotecan (irinotecan), and combinations thereof.

In another embodiment of the invention, one or more other anti-cancer treatments or agents are administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intranasally, intrapulmonary, intrathecally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, stereotactically, orally, or by direct injection or infusion.

In a preferred embodiment of the invention, the checkpoint inhibitor-refractory patient exhibits a congenital (primary) resistance to the checkpoint inhibitor treatment or an acquired (secondary) resistance to the checkpoint inhibitor treatment.

In a preferred embodiment of the invention, a checkpoint inhibitor-refractory patient exhibits a congenital (primary) resistance to treatment with a checkpoint inhibitor, manifested by a lack of response or an insufficient response to treatment with said checkpoint inhibitor for at least about 8 weeks or 12 weeks from the start of the first dose.

In a preferred embodiment of the invention, a checkpoint inhibitor-refractory patient exhibits acquired (secondary) resistance to checkpoint inhibitor therapy as evidenced by an initial response to the checkpoint inhibitor therapy, but a subsequent recurrence and progression of one or more tumors.

In yet another preferred embodiment of the invention, the checkpoint inhibitor-refractory patient exhibits congenital (primary) resistance or acquired (secondary) resistance to treatment with one or more of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, LAG-3 inhibitors.

In one embodiment of the invention, the checkpoint inhibition therapy and/or co-stimulation checkpoint therapy is synergistic with mycobacteria.

In one embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from: cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (Fab), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable region fragments (scFv), disulfide stabilized variable region fragments (dsFv), or other antigen binding fragments thereof, directed against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcCR 3, and combinations thereof; and (ii) an inactive whole cell mycobacterium, wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors or inactive whole cell mycobacteria alone, optionally wherein checkpoint inhibition therapy comprises administration of a sub-therapeutic amount and/or duration of the one or more blockers.

In one embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from: a cell, protein, peptide, antibody, ADC (antibody-drug conjugate), Fab fragment (Fab), F (ab')2 fragment, diabody, triabody, tetrabody, precursor, single chain variable fragment (scFv), disulfide stabilized variable region fragment (dsFv), or other antigen binding fragment thereof, and combinations thereof, directed against CTLA-4, PD-1, or PD-L1; and (ii) inactive whole cell mycobacteria.

In one embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors selected from: ipilimumab, nivolumizumab, pembrolizumab, azetolizumab, duvatuzumab, tremelimumab, sibradizumab, avelumab, certralizumab, terilisib, MGA012, MGD013, MGD019, entilizumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlinab (BMS986016), LAG525, IMP321, REGN2810 (cimirapril mab), REGN3767, pexidatinib, LY 2855, FPA008, BLZ 302945, GDC0919, empatab, indoximid, BMS986205, CPI-444, MEDI9447, PBF509, liriluzumab, and combinations thereof, and (ii) inactive whole cell mycobacteria.

In a preferred embodiment of the invention, the one or more checkpoint inhibitors are selected from ipilimumab and/or nivolumab.

In one embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in a subject refractory to a checkpoint inhibitor, wherein the one or more tumors are associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, skin cancer and soft tissue sarcoma, preferably wherein the one or more tumors are associated with pancreatic cancer, colorectal cancer, prostate cancer, skin cancer or ovarian cancer.

In another embodiment of the invention, there is provided a method of treating, reducing, inhibiting or controlling a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors, (ii) inactive whole-cell mycobacteria, and (iii) a co-stimulatory checkpoint treatment performed simultaneously, separately or sequentially with administration of the mycobacteria, wherein the co-stimulatory checkpoint treatment comprises administration of one or more binding agents selected from the group consisting of cells, proteins, peptides, antibodies, ADCs (antibody-drug conjugates), Fab fragments (fabs), F (ab')2 fragments, diabodies, triabodies, tetrabodies, precursors, single chain variable fragments (scfvs), disulfide stabilized variable region fragments (dsfvs) or other antigen binding fragments thereof directed to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, and combinations thereof; wherein the method results in an enhanced therapeutic effect relative to administration of one or more checkpoint inhibitors, co-stimulatory checkpoint therapy or inactive whole cell mycobacteria alone, and optionally, the co-stimulatory checkpoint therapy comprises administration of a sub-therapeutic amount and/or duration of the binding agent.

In another embodiment of the invention, the co-stimulation checkpoint treatment comprises administering one or more binding agents selected from the group consisting of utolimumab, urelimumab, MOXR0916, PF04518600, MEDI0562, GSK3174988, MEDI6469, RO7009789, CP870893, BMS986156, GWN323, JTX-2011, varlizumab, MK-4166, NKT-214, and combinations thereof.

In another embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors, (ii) inactive whole cell mycobacteria, and (iii) a co-stimulatory checkpoint treatment performed simultaneously, separately or sequentially with the administration of the mycobacteria, wherein the co-stimulatory checkpoint treatment comprises administration of one or more binding agents, wherein the binding agent is an agonistic antibody, optionally wherein the method comprises administration of a sub-therapeutic amount and/or duration of the co-stimulatory checkpoint binding agent.

In yet another embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the checkpoint inhibition therapy comprises administration of two or more blockers selected from the group consisting of cells, proteins, peptides, antibodies, ADC (drug-drug conjugate) antibodies against CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, DcR3, and combinations thereof, A Fab fragment (Fab), F (ab')2 fragment, diabody, triabody, tetrabody, precursor, single chain variable fragment (scFv), disulfide stabilized variable region fragment (dsFv), or other antigen binding fragment thereof, and optionally wherein the checkpoint inhibition therapy comprises administration of a sub-therapeutic amount and/or duration of the blocking agent.

In yet another embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or controlling one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, wherein the checkpoint inhibition therapy comprises administration of two or more blockers, wherein the two or more blockers are directed against any one of the following combinations: CTLA-4 and PD-1, CTLA-4 and PD-L1, PD-1 and LAG-3, or PD-1 and PD-L1.

Suitable specific combinations include: duvalirudumab + tremelimumab, Nabrireulimumab + Epipilimumab, pembrolizumab + Epipilimumab, MEDI0680+ Duvalirudumab, PDR001+ FAZ053, Nabrireulimumab + TSR022, PDR001+ MBG453, Nabrireulimumab + BMS986016, PDR001+ LAG525b, pembrolizumab + IMP321, REGN2810 (Simimipril mab) + REGN3767, and other suitable combinations.

In one embodiment of the invention, there is provided an inactive whole cell mycobacterium for use in treating, reducing, inhibiting or managing one or more tumors in a checkpoint inhibitor-refractory patient, wherein the checkpoint inhibitor-refractory patient is intended for checkpoint inhibition therapy simultaneously, separately or sequentially with administration of the mycobacterium, further comprising co-stimulatory checkpoint therapy simultaneously, separately or sequentially with administration of the mycobacterium, directly against any one of the following combinations: CTLA-4 and CD40, CTLA-4 and OX40, CTLA-4 and IDO, OX-40 and PD-L1, PD-1 and OX-40, CD27 and PD-L1, PD-1 and CD137, PD-L1 and CD137, OX-40 and CD137, CTLA-4 and IDO, PD-1 and IDO, PD-L1 and IDO, PD! And A2AR, PD-L1 and A2AR, PD1 and GITR, PD-L1 and GITR, PD1 and ICOS, PD-L1 and ICOS, PD1 and CD27, PD-L1 and CD27, PD1 and CD122, PD-L1 and CD122, PD1 and CSF1R, PD-L1 and CSF1R, and other such suitable combinations.

Suitable specific combinations include: avermentizumab + utomolizumab, Naviuliuzumab + urelumab, pembrolizumab + utomolizumab, Atezolimumab + MOXR0916 + -bevacizumab (bevacizumab), Avermentizumab + PF-04518600, Duviuzumab + MEDI0562, pembrolizumab + GSK3174998, Trimetuzumab + Duvizukiumab + MEDIA + MEDI6469, Trimetuzumab + MEDI0562, Utouuzumab + PF-04518600, Atezolimumab + RO7009789, Trimetuzumab + CP870893, Naviuliuzumab + 6156, PDR001+ N323, Naviuiuzumab + JTX-2011, Atezolizumab + GDC0919, Peviitumumab + Epadoxystatab, Epimetriuzumab + GWrivuzumab, GWthiolazetamab + GWVTaB 6205, GWVTaD-2835, GWvettazetamab + GWrit + PDX-2011, Atezoliuzumab + GDC0919, Poviuzumab + EPITaP-94E + E-PDK-D-III, GWrituzumab + GWoclavavizumab, GWoclavavivaX-33509, GWoclavavivaX + GWrit + G-D-3, GWrit + E-D-E + E-D-E-D-E-D-E + E-D-E-, PDR001+ BLZ945, tremelimumab + LY 3022855.

In another embodiment of the invention, there is provided a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in an individual refractory to a checkpoint inhibitor, wherein the method comprises administering to the individual simultaneously, separately or sequentially: (i) one or more checkpoint inhibitors, (ii) an inactive whole cell mycobacterium, and (iii) a costimulatory checkpoint treatment simultaneously, separately or sequentially with administration of the mycobacterium, further comprising administration of one or more other anti-cancer treatments or agents, wherein the method results in an enhanced therapeutic effect relative to administration of the one or more checkpoint inhibitors, the costimulatory checkpoint treatment, the one or more other anti-cancer treatments or agents, or the inactive whole cell mycobacterium alone.

In one embodiment or method of the invention, the one or more additional anti-cancer treatments or agents are selected from: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, such as metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, such as OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, such as GVAX or CIMAvax.

In another method of the invention, the anti-cancer treatment is selected from: microwave radiation, radiofrequency ablation, targeted radiotherapy such as stereotactic ablation radiotherapy (SABR), embolization, cryotherapy, ultrasound, high intensity focused ultrasound, radio knife, hyperthermia, cryoablation, electrotome heating, hot water injection, alcohol injection, embolization, radiation exposure, photodynamic therapy, laser beam exposure, and combinations thereof.

In one embodiment or method of the invention, the TLR agonist comprises Mepact, Krestin (PSK), MRx0518(4D Pharma), IMO-2125(tilsotolimod), CMP-001, MGN-1703(lefitolimod), entolimod, SD-101, GS-9620, imiquimod, resiquimod, MEDI4736, poly I: C, CPG7909, DSP-0509, VTX-2337(motolimod), MEDI9197, NKTR-262, G100, or PF-3512676, and combinations thereof.

Suitable specific combinations include: ipilimumab + MGN1703, pembrolizumab + CMP001, pembrolizumab + SD101, tremelimumab + PF-3512676, resiquimod + pembrolizumab (pembrolizumab).

In one embodiment or method of the invention, chemotherapy comprises the administration of one or more agents selected from the group consisting of: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mechlorethamine, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, capecitabine, leucovorin, carboplatin, oxaliplatin, gemcitabine, folfinirox, paclitaxel, pemetrexed, irinotecan, and combinations thereof.

In one embodiment or method of the invention, one or more additional anti-cancer treatments or agents are administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intranasally, intrapulmonary, intrathecally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, stereotactically, orally, or by direct injection or infusion.

In one embodiment or method of the invention, the neoplasia, tumor or cancer is associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, malignant epithelial tumors, head and neck cancer, skin cancer and soft tissue sarcoma, preferably wherein the neoplasia, tumor or cancer is associated with pancreatic gland, colorectal cancer, prostate cancer, skin cancer or ovarian cancer, optionally wherein the neoplasia, tumor or cancer is metastatic.

In another preferred embodiment or method of the invention, the neoplasia, tumor or cancer is associated with a sarcoma, preferably a soft tissue sarcoma or a non-soft tissue sarcoma. Particularly preferred non-soft tissue sarcomas include osteosarcoma (osteosarcoma), Ewing's sarcoma) and chondrosarcoma. Particularly preferred sarcomas include undifferentiated sarcoma of pleomorphism (UPS), angiosarcoma, smooth muscle sarcoma, dedifferentiated liposarcoma (DDL), synovial sarcoma, rhabdomyosarcoma, epithelioid sarcoma, mucinous myosarcoma, alveolar soft tissue sarcoma, parachordoma/myoepithelioma (parachlorosarcoma/myoepilioma), liposarcoma of pler type, extraosseous mucinous chondrosarcoma, malignant sphingoid tumors (malignant periphytol new skin tumors). The patient may be younger than 50 years, or younger than 20 to 30 years, or a teenager (teenager) or adolescent (<16 years) or a child (0-14 years). Alternatively, one or more of the sarcoma tumors exhibit increased staining/expression of PD-L1 or PD-1. Optionally, the inactive whole cell mycobacterium and/or checkpoint inhibitor and/or co-stimulatory binding agent is administered by intratumoral, peritumoral, perilesional or intralesional administration.

In one embodiment or method of the invention, the inactive whole cell mycobacterium is selected from the group consisting of: mycobacterium vaccae, Mycobacterium obuense, Mycobacterium paratacticum, Mycobacterium aureofaciens, M.indicus pranii, Mycobacterium phlei, and combinations thereof, optionally in unprocessed form. Preferably, the inactive whole cell mycobacterium is mycobacterium obuense.

In one embodiment or method of the invention, the inactive whole-cell mycobacterium and/or checkpoint inhibitor and/or co-stimulatory binding agent is administered by a parenteral, oral, sublingual, nasal or pulmonary route, preferably the parenteral route is selected from subcutaneous (subeutaneous), intradermal, subdermal (subdermal), intraperitoneal or intravenous, intratumoral, peritumoral, perilesional or intralesional administration.

In one embodiment or method of the invention, the inactive whole cell mycobacteria is at about 10⁴To about 10¹⁰A cell, preferably about 10⁷To about 10⁹The amount of individual cells is administered.

In further embodiments, the methods of the invention comprise one or more of: 1) reducing or inhibiting growth, proliferation, migration, or invasion of tumor or cancer cells in which metastasis may or does occur, 2) reducing or inhibiting formation or establishment of metastasis to one or more other sites, locations, or regions distinct from the primary tumor or cancer caused by the primary tumor or cancer, 3) reducing or inhibiting growth or proliferation of metastasis to one or more other sites, locations, or regions distinct from the primary tumor or cancer after metastasis has formed or has been established, 4) reducing or inhibiting formation or establishment of other metastases after metastasis has formed or has been established, 5) extending overall survival, 6) extending progression-free survival, 7) disease stabilization, 8) improving quality of life.

In further embodiments, the methods of the invention result in enhanced therapeutic efficacy, optionally as defined by RECIST1.1, as measured by reduction or stabilization of tumor size of one or more of said tumors, including disease Stabilization (SD), Complete Remission (CR) or Partial Remission (PR) of the tumor of interest; and/or Stable Disease (SD) or Complete Remission (CR) of one or more non-target tumors.

In one embodiment or method of the invention, the checkpoint inhibitor-refractory patient exhibits either a congenital (primary) resistance to treatment with the checkpoint inhibitor or acquired.

However, the benefit or improvement of treatment is not necessarily a cure or complete destruction of all target proliferating cells (e.g., neoplasia, tumor or cancer or metastasis), nor an ablation of all pathologies, adverse symptoms or complications or hyperproliferative cellular diseases associated with or caused by cell proliferation, such as neoplasia, tumor or cancer or metastasis. For example, by inhibiting the progression or progression of a tumor or cancer, partial destruction of a tumor or cancer cell mass, or stabilization of tumor or cancer cell mass, size, or cell number, mortality can be reduced and longevity extended even if left for only days, weeks, or months, even if some or most of the tumor or cancer mass, size, or cells are still present.

Specific non-limiting examples of therapeutic benefit include a reduction in the volume (size or cell mass) or number of cells of a neoplasia, tumor or cancer or metastasis, an inhibition or prevention of an increase in the volume (e.g., stabilization) of a neoplasia, tumor or cancer, a slowing or inhibition of progression, worsening or metastasis of a neoplasia, tumor or cancer, or an inhibition of proliferation, growth or metastasis of a neoplasia, tumor or cancer.

In one embodiment of the invention, the combinations and methods disclosed herein provide a detectable or measurable improvement or overall response in terms of irRC (derived from time point efficacy assessment and based on tumor burden), including one or more of: (i) irCR-complete disappearance of all lesions, whether measurable or not, and no new lesions (by repeated recording, continuous assessment, should not be less than 4 weeks from the date of first recording); (ii) irPR-reduced tumor burden ≧ 50% relative to baseline (confirmed by serial assessment at least 4 weeks after first recording).

The inventive method may not be immediately effective. For example, an increase in the number or mass of neoplastic, tumor or cancer cells can be performed after treatment, but over time, a subsequent stabilization or decrease in tumor cell mass, size or cell number ultimately occurs in a given individual.

In one embodiment of the invention, the combinations and methods disclosed herein result in a clinically relevant improvement in one or more markers of disease status and progression selected from one or more of: (i) the method comprises the following steps Overall survival, (ii): progression-free survival, (iii): overall remission rate, (iv): reduced metastatic disease, (v): circulating levels of tumor antigens, such as carbohydrate antigen 19.9(CA19.9) and carcinoembryonic antigen (CEA) or other tumor-dependent antigens, (vii) nutritional status (body weight, appetite, serum albumin), (viii): control of pain or analgesic use, (ix): CRP/albumin ratio.

In another embodiment, the checkpoint inhibition therapy comprises administering a blocking agent, wherein the blocking agent is an antibody selected from the group consisting of: AMP-224(Amplimmune, Inc), BMS-986016, or MGA-271, and combinations thereof. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

BMS-986016 is a human LAG-3 specific fully human antibody isolated from an immunized transgenic mouse expressing human immunoglobulin genes. It is expressed as an IgG4 isotype antibody that includes a stabilizing hinge mutation that attenuates Fc receptor binding (S228P) to reduce or eliminate the potential for antibody or complement-mediated killing of target cells. The heavy and light chain amino acid sequences of BMS-986016 are provided in SEQ ID NOs 17 and 18 of WO 2015/042246.

In another embodiment, the checkpoint inhibition therapy comprises administration of BMS-986016 intravenously at a dose of about 20mg to about 8000mg once every two weeks, optionally up to forty-eight infusions.

In another embodiment, the checkpoint inhibition therapy comprises administration of a blocking agent, wherein the blocking agent is an antibody that specifically binds B7-H3, such as entilizumab, an engineered Fc humanized IgG1 monoclonal antibody directed against B7-H3, with potent anti-tumor activity (macrogenetics, Inc.); or MGD009, a B7-H3 amphipathic reorientation (DART) protein, can bind to CD3 on T cells and B7-H3 on target cells and has been found to recruit T cells to the tumor site and promote tumor eradication, or MGD009 is a humanized DART protein. MGC018 is an anti-B7-H3 Antibody Drug Conjugate (ADC) with a Duocarmycin load and a cleavable peptide linker.

In some embodiments, the checkpoint inhibition therapy comprises administration of an anti-B7-H3 binding protein selected from the group consisting of: DS-5573(Daiichi Sankyo, Inc.), Enbilizumab (Macrogenics, Inc.), and omburtamab [8H9] (Y-mabs Therapeutics, Inc) (a B7-H3 antibody labeled with radioiodine (I-131)).

In some embodiments, checkpoint inhibition therapy includes administration of indoleamine-2, 3-dioxygenase (IDO) inhibitors, such as D1-methyl-tryptophan (Lunate) and other compounds described in U.S. patent No. 7,799,776, the contents of which are incorporated herein by reference.

In certain embodiments, a co-stimulatory checkpoint therapy upregulates the cellular immune system, wherein the co-stimulatory checkpoint therapy comprises administration of a binding agent selected from the group consisting of: cells, proteins, peptides, antibodies or antigen-binding fragments thereof directed against CD27, OX40, GITR, or CD137, and combinations thereof, such as CD137 agonists, including but not limited to BMS-663513 (urellumab, anti-CD 137 humanized monoclonal antibody agonist, Bristol-Myers Squibb); agonists of CD40, such as CP-870, 893 (a-CD 40 humanized monoclonal antibody, Pfizer); OX40(CD134) agonists (e.g., anti-OX 40 humanized monoclonal antibodies, AgonOx and those described in U.S. patent No. 7,959,925) and Astra Zeneca's MEDI0562, a humanized OX40 agonist; MEDI6469, murine OX4 agonist; and MEDI6383, an OX40 agonist; or an agonist of CD27, such as CDX-1127 (a-CD 27 humanized monoclonal antibody, Celldex). Suitable anti-GITR antibodies include TRX518(Tolerx), MK-1248(Merck), CK-302, and suitable anti-4-1 BB antibodies for use in the invention include PF-5082566 (Pfizer).

TIGIT is a checkpoint receptor believed to be involved in mediating tumor T cell failure. TIGIT has been shown to be independent of other checkpoint molecules CTLA-4 and PD-1, and associated with NK cell depletion in tumor-bearing mice and colon cancer patients. Blocking with TIGIT prevented NK cell failure and enhanced NK cell-dependent tumor immunity in various tumor-bearing mouse models. Furthermore, blockade of TIGIT produced potent tumor-specific T cell immunity in an NK cell-dependent manner, enhanced therapeutic efficacy with PD-1 ligand PD-L1 antibody, and sustained immunological memory in tumor re-challenge models.

There is evidence that greater infiltration of MDSCs, including CD68+ or CD163+ specific tumor-associated macrophages (TAMs), is associated with checkpoint inhibitor resistance. In vivo studies also indicate that inhibition of CD103+ DC recruitment by β -catenin signaling results in primary resistance.

The loss of β 2 microglobulin (B2M) is a mechanism of acquired resistance to immunotherapy that results from insufficient antigen presentation. Loss of B2M interferes with MHC class I heavy chain folding, resulting in loss of its receptor localization and disruption of downstream signaling that would otherwise propagate T cell activation and recruitment. Tumor down-regulation of MHC class I molecules is another mechanism of tumor immune escape, nullifying anti-tumor T cell responses.

The function of APC in anti-tumor immunity is to metastasize tumor antigens to tumor-draining lymph nodes for tumor-specific CD8+ T cell priming. In melanoma, CD103+ DC is the only APC with this function. In a melanoma mouse model, administration of the growth factors FLT3L and poly I: C had expanded and activated CD103+ DC progenitors in the tumor, reversing resistance to PD-L1. Furthermore, studies have shown that in non-inflamed tumors, failure of accumulation of CD103+ dendritic cells (this cell type is the major source of the T cell recruitment chemokine CXCL 9/10) leads to insufficient entry of therapeutically activated T cells and immunotherapy resistance. Thus, the absence of CD103+ DC in the tumor microenvironment may be the primary mechanism for resistance to multiple immunotherapies.

Conventional dendritic cells (cdcs) are specialized antigen presenting cells that control T cell immunity. Pedigree-tracking experiments in mice have mapped two developmentally and functionally distinct populations of cDC1 and cDC2, located in peripheral tissues, defined by the expression of CD103 and CD11b, respectively. These lineages and their functions are conserved among humans. Of these, cDC1 is highly efficient at cross-presenting antigens to cytotoxic T cells and is the main stimulatory population of cdcs within the tumor, both to generate anti-tumor immunity in draining Lymph Nodes (LNs) and to interact directly with effector T cells in the tumor microenvironment. Furthermore, cDC1 is critical for checkpoint blockade of therapeutic response.

TIM-3 is highly expressed in intratumoral CD103+ dendritic cells, and administration of TIM-3 antibodies indirectly enhances CD8+ T cell responses during chemotherapy.

TLR3 agonist polyribose creatine: polyribonic acids (poly I: C) induce type I IFN production and DC maturation. CD141+ DC is the human equivalent of murine CD8+/CD103+ DC, and TLR3 and TLR8 are expressed by CD141+ DC. Injection of TLR3 and a TLR7 agonist (resiquimod) into mice resulted in co-upregulation of the co-stimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c + DCs.

The term "combination" as used throughout the specification is meant to include administration of the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent simultaneously, separately or sequentially with administration of the mycobacterium. Thus, the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent and the mycobacterium may be present in the same or separate pharmaceutical preparations and may be administered simultaneously or at different times.

Thus, inactive whole cell mycobacteria and checkpoint inhibitors and/or costimulatory checkpoint binding agents may be provided as separate medicaments for administration simultaneously or at different times.

Preferably, the inactive whole cell mycobacterium and the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent are provided as separate medicaments for administration at different times. When administered separately and at different times, inactive whole-cell mycobacteria or checkpoint inhibitors and/or co-stimulatory checkpoint binding agents may be administered first; however, it is suitable to administer the checkpoint inhibitor and/or costimulatory checkpoint binding agent first, followed by administration of the inactive whole cell mycobacterium. In addition, both may be administered on the same day or on different days, and may be administered on the same schedule or on different schedules during the treatment cycle.

In one embodiment of the invention, the treatment cycle consists of administering the inactive whole cell mycobacterium daily, weekly, biweekly or monthly, while administering the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent weekly or biweekly, every three weeks or four weeks or longer. Alternatively, the inactive whole cell mycobacterium is administered before and/or after administration of the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent.

In another embodiment of the invention, the inactive whole cell mycobacterium is administered to the patient before and after administration of the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent. That is, in one embodiment, whole cell non-pathogenic heat-inactivated mycobacteria are administered to a patient before and after the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent.

In another embodiment of the invention, the inactive whole cell mycobacterium is administered to the patient before and after administration of the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent and/or one or more other anti-cancer therapies or agents, including: adoptive cell therapy, surgical therapy, chemotherapy, radiotherapy, hormonal therapy, small molecule therapy, such as metformin, receptor kinase inhibitor therapy, hyperthermia, phototherapy, radioablation therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, biological therapy, HDAC inhibitors, such as OKI-179, BRAF inhibitors, MEK inhibitors, EGFR inhibitors, VEGF inhibitors, P13K δ inhibitors, PARP inhibitors, mTOR inhibitors, hypomethylating agents, oncolytic viruses, TLR agonists including TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, such as MRx0518(4D Pharma), STING agonists (including MIW815 and SYNB1891) and cancer vaccines, such as GVAX or CIMAvax.

Dose delay and/or dose reduction and schedule adjustments are performed as needed depending on the tolerance of the individual patient to the treatment.

Alternatively, administration of the checkpoint inhibitor and/or co-stimulatory checkpoint binding agent can be performed simultaneously with administration of an effective amount of inactive whole cell mycobacterium.

The individual receiving checkpoint inhibition therapy and/or co-stimulation checkpoint therapy according to the present invention may be administered simultaneously, separately or sequentially with the administration of the inactive whole cell mycobacterium.

In one aspect of the invention, an effective amount of an inactive whole cell mycobacterium may be administered as a single dose. Alternatively, an effective amount of inactive whole cell mycobacteria may be administered in multiple (repeated) doses, e.g. more than two, more than three, more than four, more than five, more than ten or more than twenty repeated doses. In multiple doses of mycobacterium administered, there may be periods of 1 week, 2 weeks, 3 weeks, 4 weeks, or combinations between the above doses.

The inactive whole cell mycobacterium may be administered between about 8 weeks, 6 weeks, or 4 weeks and/or between about 1 day, e.g., between about 4 weeks and 1 week, or between about 3 weeks and 2 weeks prior to the checkpoint inhibition treatment. Administration may be in a single dose or more preferably in multiple doses.

In one embodiment of the present invention, the inactive whole cell mycobacterium may be in the form of a drug that is administered to the patient in a dosage form.

In some cases, the container according to the invention may be a vial, ampoule, syringe, capsule, tablet or tube. In some cases, the mycobacteria may be lyophilized and formulated for resuspension prior to administration. However, in other cases, the mycobacterium is suspended in a volume of pharmaceutically acceptable liquid. In some most preferred embodiments, a container is provided comprising a single unit dose of a mycobacterium suspended in a pharmaceutically acceptable carrier, wherein the unit dose comprises about1×10⁶To about 1X10¹⁰An organism. In some very specific embodiments, the liquid comprising suspended mycobacteria is provided in a volume of from about 0.01ml to 10ml, or from about 0.03ml to 2ml, or from about 0.1ml to 1 ml. The foregoing compositions provide desirable units for the immunotherapeutic applications described herein.

Embodiments discussed in the context of the methods and/or compositions of the present invention may be used with respect to any other method or composition described herein. Thus, embodiments relating to one method or composition may also be applied to other methods and compositions of the present invention.

In some cases, the inactive whole cell mycobacterium is administered to a specific site on or in the individual. For example, the mycobacterial compositions of the invention (such as those comprising mycobacterium obuense, in particular) may be administered adjacent to a tumor or adjacent to a lymph node (such as those draining tissue surrounding a tumor). Thus, in some cases, the local administration of the mycobacterial composition may be in the vicinity of a lymph node on the posterior neck, tonsil, axilla, groin, anterior neck, lower jaw, submental, or clavicle.

Inactive whole cell mycobacteria may be administered for the duration of time that a cancer or tumor is present in a patient, or until the cancer has regressed or stabilized. Whole cell non-pathogenic heat-inactivated mycobacteria may also be continued to be administered to the patient if the cancer or tumor has regressed or stabilized.

The mycobacterial compositions of the present invention will comprise an effective amount of the mycobacteria, typically dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal (e.g., a human, if appropriate). In view of the present disclosure, preparations of Pharmaceutical compositions containing mycobacteria will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences,18th ed. Further, for animal (e.g., human) administration, it is understood that the preparation should meet sterility, thermogenesis, general safety and purity standards. Specific examples of pharmacologically acceptable carriers as described herein are borate buffers or sterile saline solution (0.9% NaCl).

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gelling agents, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences,18th ed. mack Printing Company,1990, pp. 1289-1329).

In a preferred embodiment, the whole cell non-pathogenic heat-inactivated mycobacterium is administered via a parenteral route selected from the group consisting of: subcutaneous, intradermal, subdermal, intraperitoneal, intravenous, and intravesicular injections. Intradermal injection can deliver an intact portion of the mycobacterial composition to the dermis layer accessible for immune surveillance and thus can select an anti-cancer immune response at the regional lymph nodes and promote immune cell proliferation.

Although in a highly preferred embodiment of the invention, the mycobacterial composition is administered by direct intradermal injection, it is contemplated that other methods of administration may be used in some circumstances.

In certain instances, a whole-cell non-pathogenic heat-inactivated mycobacterium of the present invention can be administered by injection, infusion, continuous infusion, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intravitreal, intravaginal, intrarectal, topical, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intracapsular, mucosal, intrapericardial, intraorbital, intraocular, intraoral, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, topical (topically), topical (locally), inhalation (e.g., aerosol inhalation), via catheter, via lavage, or via other methods, or any combination of the foregoing, as known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences,18th ed.

In another embodiment, the immune modulator is administered into the skin of a checkpoint inhibitor refractory patient via a microneedle device comprising a plurality of microneedles.

Table 1 below gives various methods and formulation methods for manufacturing solid microneedles according to the present invention.

Table 2 below gives a selection of microneedle device technologies for use in accordance with the present invention, which patents and patent applications are incorporated herein by reference.

Other preferred microneedle devices for use according to the present invention include: north Carolina State University (North Carolina State University) (as described in WO 2017/151727), Debioject microneedles (Debiotech, Switzerland), Microject 600(NaoPas, Israel, as described in WO 2008/047359), Nanopatch (Vaxxas, USA), SOFUSA (Kimberly-Clark, USA, as described in WO2017/189259 and WO 2017/189258), Micron Biomedical's dissolution microarray and MIMIX dissolution, controlled release microarray (Vaxess, USA).

In one embodiment, the present invention provides an immunomodulatory agent for use in treating, reducing, inhibiting or controlling cancer in an individual, wherein the immunomodulatory agent comprises a whole cell, inactive mycobacterium, and wherein the immunomodulatory agent is to be administered into the skin of the individual by a microneedle device comprising a plurality of microneedles.

In one embodiment, the microneedles are hollow. In a separate embodiment, the microneedles are solid.

In another embodiment, the plurality of microneedles are arranged in a line, square, circle, grid, or array.

In another embodiment, the microneedle device comprises 2 to 2000 microneedles per square centimeter, such as 4 to 1500 microneedles per square centimeter, or 10 to 1000 microneedles per square centimeter.

In another embodiment, the microneedles are 2-2000 microns in length, such as 20-1000 microns, or 50-500 microns, or 100-400 microns.

In another embodiment, the microneedle is configured to deliver the immunomodulatory agent intradermally, optionally wherein the immunomodulatory agent is delivered to a lymphatic vessel.

In another embodiment, the immunomodulatory agent is coated on or embedded in at least a portion of a microneedle, optionally wherein the microneedle is implanted into or removed from the skin. Preferably, the coating or microneedles are dissolvable upon contact with the skin.

In another embodiment, wherein the microneedle is hollow and the immunomodulator is delivered intradermally by the microneedle in the form of a suspension, optionally wherein the microneedle is implanted in or removed from the skin.

In another embodiment, a method of treating, reducing, inhibiting or managing a neoplasia, tumor or cancer in a patient refractory to a checkpoint inhibitor is provided, wherein the method comprises:

(i) there is provided a microneedle device comprising a plurality of microneedles,

(ii) causing the microneedles to penetrate the skin of the individual and assume an anchored state in which the microneedles are anchored in the skin and extend from the microneedle device,

(iii) delivering an amount of an immunomodulatory agent into the skin through the microneedle, wherein the immunomodulatory agent comprises whole cell, inactive mycobacteria.

In another embodiment, a kit of parts for delivering at least one immunomodulator into the skin of a checkpoint inhibitor refractory patient is provided (kit of parts) comprising:

a microneedle device comprising a plurality of microneedles, and

one or more immune modulators selected from the group consisting of:

whole-cell inactive mycobacteria such as Mycobacterium vaccae such as NCTC 11569, Mycobacterium obuense such as NCTC 13365, Mycobacterium parafortuitum, Mycobacterium aurum, M.indicus pranii, Mycobacterium phlei, and combinations thereof, and;

a checkpoint inhibitor selected from a cell, protein, peptide, antibody or antigen-binding fragment thereof against CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7-H3, B7-H4, B7-H6, A2AR, or IDO, and combinations thereof.

In another embodiment, a microneedle device is provided that comprises a plurality of microneedles and a composition comprising whole cell inactive mycobacteria thereon or therein.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and immunology or related fields are intended to be within the scope of the following claims.

The invention is further described with reference to the following non-limiting examples.

Example 1

Use of 10 from pancreatic cancer cell lines obtained from KPC mice⁵The individual cells were injected subcutaneously in the flank into adult C57BL/6 mice (Hingorani et al cancer Cell,2005,7: 469-48). These murine pancreatic cancer cells carry mutations in Kras, p53 and Pdx-Cre (Hingorani et al cancer Cell,2005,7: 469-48).

When the injected tumor cells had grown to a palpable tumor (day 0), mice were left untreated or received the following treatments:

1)0.1mg of heat-inactivated whole cell mycobacterium obuense NTCT 13365/mouse, injected subcutaneously on alternate days, alternating dorsum cervicodynia and caudad basics, at 2 days intervals over the length of study, over 5 days;

2) injecting 10mg/kg anti-PDL-1 monoclonal antibody intraperitoneally once a week;

3) anti-PDL-1 and mycobacterium obuense NTCT 13365 were combined at the doses and schedules described above for the two compounds used alone.

Tumor growth was monitored throughout the study to determine if the administered treatment had the effect of reducing tumor size and improving the survival prospects.

The data shown in figure 1 demonstrate that mice receiving a therapeutic combination of anti-PDL-1 and mycobacterium obuense NTCT 13365 exhibit a sustained reduction in tumor size and appear to control tumors. This reduction in tumor size was more pronounced compared to mice receiving either treatment alone. Tumor growth in untreated mice is uncontrolled and rapidly dies of the disease.

Example 2

The effect of combined treatment with IMM-101 (heat-inactivated whole cell Mycobacterium obuense NTCT 13365) and checkpoint inhibitors was studied in C57BL/6 mice bearing subcutaneous checkpoint resistant B16-F10 tumors. Mice were transplanted at D0. Mice were randomized at D1, individually at D1, D3, D5, D7, D9, D11, D13 and D15 (half of surviving mice) or D16 (half of surviving mice) (Q2Dx8) received a total of 8 subcutaneous injections of IMM-101 at 0.1 mg/mouse or individually at 10mg/kg a total of 4 intraperitoneal injections of anti-PD 1 or anti-CTLA 4 (twice a week, two consecutive weeks at D1, D5, D8 and D12: TWx2) or a combination of both. On days 15 and 16, half of all surviving mice were killed after the last treatment (terminated) and tumor immune infiltrating cells and spleen immune cells (ratio of CD8+ cells to FoxP3Treg cells) were then quantified by FACS analysis (fig. 3). It can be seen that the ratio of CD8+ to tregs is increased, which translates into an enhanced tumor regression effect in human checkpoint refractory patients.

Example 3

Will be 1x10⁶One EMT-6 mouse mammary tumor cell was injected subcutaneously into BALB/C mice. The average volume of the tumor reaches 80-120mm²Treatment was started on day (about day 7) using IMM-101 at 0.1 mg/mouse per day, anti-PD-1 at 10 mg/kg/injection twice weekly, a combination of IMM-101 and anti-PD-1 or vehicle (FIG. 4). On day 28, mice were euthanized and tumor draining lymph nodes and spleens were removed from all mice. Tumor volumes were measured in mice every 3 days. The doubling time of the tumor size (the ratio of the tumor size to the size at the start of treatment) after treatment was measured (fig. 5) and tumor volume was plotted against time (fig. 2). Measurement of CD8 on day 28 by flow cytometry⁺T cell/FoxP 3⁺The proportion of regulatory T cells (combination of two experiments) (FIG. 6) and the proportion of IFN-. gamma./IL-10 in the spleen cell supernatant was determined by ELISA. IFN-. gamma./IL-10 ratios (FIG. 7) (. p) were measured by ELISA in splenocyte supernatants stimulated with anti-CD 3 for 72 hours on day 28<0.05，**p<0.01，***p<0.001). It can be seen that an increased ratio of CD8+ to tregs and an increased ratio of IFN- γ/IL-10 will translate into an enhanced tumor regression effect in human patients.

Example 4

IMM-101 (300. mu.g) was injected subcutaneously plantar to C57BL/6WT or Batf 3-/-mice. Drained LN was harvested 7 days later and re-stimulated with either IMM-101 alone or culture medium for 72 hours. A) Schematic of the experimental procedure, B) after restimulation, IFN-g levels in supernatants were measured by ELISA. The Baft 3-/-mice lost the ability of IMM-101 to induce IFN- γ secretion in vivo, indicating that CD103+ dendritic cells were required in this pathway (figure 8).

Example 5

A study has been carried out to study the effect of a preparation of heat-inactivated whole cell mycobacterium obuense (IMM-101) in patients with previously treated colorectal cancer, in combination with a study of the safety and efficacy of treatment with IMM 101 in combination with checkpoint inhibitors in patients with advanced melanoma radiation-induced immunogenic tumor necrosis.

Treated patients presenting unresectable stage III or IV metastatic melanoma had not previously been treated (group a) or had developed disease during PD-1 blockade (group B).

This study was aimed at studying whether the combination of IMM-101 and nivolumab is well tolerated and to study the efficacy signals of this combination in untreated patients (group a) and patients whose disease had progressed during PD-1 blockade (group B-checkpoint refractory/resistant patients).

IMM-101 was injected into the skin above the deltoid muscle in a single 0.1mL intradermal injection of IMM 101(10mg/mL), with the arm alternating between doses. The investigator will have previously received appropriate training for the intradermal injection technique.

Previous clinical experience with IMM-101 showed that this dose was safe and well tolerated. The skin reaction that occurs at the injection site is characterized by erythema, local swelling, and occasional mild ulceration. All symptoms should be expected in view of the known pharmacological effects of the product and previous clinical experience. Furthermore, safety and tolerability studies using IMM 101 have yielded data indicating that skin reactions are satisfactorily resolved over time and do not impair daily activities.

In the study, each patient was first administered IMM-101 and then monitored for vital signs for at least 2 hours under medical supervision and used a resuscitation device as a precautionary measure.

The treatment regimen was 1 dose of IMM 101 every 2 weeks for the first 3 doses, followed by a rest of 4 weeks, followed by 1 dose every 2 weeks for the next 3 doses. This will be followed by administration every 4 weeks and allow a +/-2 day window.

Nivolumetrizumab and ipilimumab are administered according to prescription information. According to the evaluation table, patients will receive IMM-101 first when either nivolumab or ipilimumab is administered concurrently with IMM-101 on the same day. Each patient in the study was administered a first dose of nivolumetrimab at least 2 hours after the first dose of IMM-101.

For group B patients, ipilimumab can be used as a follow-up treatment with IMM-101 instead of nivolumab, as they continue to study according to RECIST1.1 and/or researchers believe that continued acceptance of nivolumab is no longer appropriate for clinical progression reasons.

Patients in both groups continued treatment until disease progression (assessed by the solid tumor efficacy assessment criteria [ RECIST ] 1.1) met the following: unacceptable side effects, investigator's decision to discontinue treatment, withdrawal of patient consent, or 18 months of IMM-101 treatment, whichever was earlier. Patients who remain in complete remission after two scanning examinations should continue treatment unless the investigator deems this to be irrelevant to the patient's greatest benefit. Patients in groups a and B with disease progression may continue to be treated with nivolumab + IMM-101 in the study if they have clinical benefit and no decline in performance and the investigator determines that the study treatment has no clinically relevant adverse effects, or is not considered to require alternative treatment.

Patients in group B did not respond to treatment with IMM-101+ nivolumab, i.e., they had recorded progression or clinical progression of RECIST1.1 (but not met RECIST1.1 rules for progressive disease), and in both cases had no previously recorded response, and if the investigator considered this to be in line with the patient's greatest benefit and the patient had not previously received a non-investigational study (monotherapy or combination therapy) of ipilimumab, the option to modify the treatment regimen in the study to ipilimumab + IMM-101 could be selected. As this treatment may continue until a maximum of 4 doses of ipilimumab are received or prematurely terminated due to unacceptable side effects, the investigator's decision to terminate the treatment, withdrawal of the patient's consent, or 18 months of IMM-101 treatment (whichever is earlier). Patients in group B who received all 4 doses of ipilimumab after this time should continue the study and follow protocol assessments. They may continue to receive IMM 101 during this period until unacceptable side effects occur, the investigator decides to terminate therapy, withdrawal of patient consent, or 18 months of IMM-101 treatment (whichever is earlier). After baseline evaluation, all patients will be evaluated for safety, response to treatment (by planning scans), and survival according to the study evaluation schedule, and given an opportunity to receive an 18-month study of IMM-101 treatment. Scans were scheduled after the first baseline, at week 12 for patients in group a and week 6 for patients in group B. Subsequent scans are every 8 weeks, and if clinically indicated, unscheduled scans can be performed, for example, to confirm progression. At the discretion of the investigator, the frequency of scanning may be increased to once every 12 weeks for patients who continue the study for more than 52 weeks.

Nivolumitumumab was administered by intravenous infusion at 3mg/kg every two weeks according to the prescription information. In the case of administration of nivolumetrizumab and IMM 101 on the same day, IMM 101 will be administered first. Each patient in the study was administered a1 st dose of nivolumab at least 2 hours after the first dose of IMM-101. The dosage may be delayed if a toxic reaction occurs.

If used in a study with group B patients, ipilimumab is administered every 3 weeks for up to 4 doses at 3mg/kg intravenous infusion over 90 minutes, according to the prescription information. The first dose of ipilimumab may begin at any time during the study, but must be at least 2 weeks after the last dose of nivolumab. In the case of ipilimumab and IMM 101 administered on the same day, IMM 101 will be administered first. The dose may be delayed if a toxic response occurs, but all ipilimumab doses must be administered within 16 weeks of the initial dose.

Patients in group B who received all 4 doses of ipilimumab after this time should continue the study and follow protocol assessment. They may continue to receive IMM 101 during this period until unacceptable side effects occur, the investigator decides to terminate treatment, withdrawal of the patient's consent, or 18 months of IMM-101 treatment (whichever is earlier).

Example 6

The effect of combined treatment with IMM-101 (heat-inactivated whole cell Mycobacterium obuense NTCT 13365) and checkpoint inhibitors was studied in C57BL/6j mice bearing subcutaneous checkpoint resistant B16-F10 (melanoma) tumors, as used in example 2. Mice were inoculated with 50,000 tumor cells, and then when the tumor volume reached 54-125mm³(average TV range of each group was 82-88mm³) Then, 10 of them are added in each groupMice were randomly grouped. Animals were dosed on and after day 0 as follows: every three days 100ul PBS (vehicle) was injected subcutaneously (group 1); every three days near the tumor [ around the tumor ]]0.1 mg/mouse IMM-101 subcutaneously (group 2); twice weekly anti-PD 1 intraperitoneal [ RMP1-14]Administration (group 3), or twice weekly anti-PD-1 [ RMP1-14 ]]And IMM-101 every 3 days (group 4; intraperitoneal and peritumoral, respectively). Mice were dosed until 3000mm due to moribund or maximal Tumor Volume (TV)³(for later).

The results are shown in FIGS. 9-14, showing mean TV +/-SE (FIG. 9), mean TV without SE (FIG. 10), mean TV without SE up to study day 16 (FIG. 11), median TV (FIG. 12), median TV up to study day 16 (FIG. 13), and Kaplan-Meier assay survival plot (FIG. 14).

Calculation of tumor growth inhibition (%) for each group and study day indicated that group 3 (anti-PD 1 only) showed a% TGI of 29.07% at study day 16, while group 4 (IMM-101 plus anti-PD-1) showed a% TGI of 52.90% at study day 16.

As can be seen from figures 9-14, the combined efficacy of IMM-101 and anti-PD 1 was significantly improved in this checkpoint refractory mouse model compared to anti-PD 1 alone, in terms of% TGI values, particularly when IMM-101 was administered subcutaneously near the tumor. Furthermore, the combination also resulted in a greater percentage of survival compared to anti-PD 1 alone.

Example 7

The effect of combined treatment with IMM-101 (heat-inactivated whole cell Mycobacterium obuense NTCT 13365) and checkpoint inhibitors was studied in C57BL/6j mice bearing subcutaneous checkpoint resistant Pan02 (pancreatic) tumors. Mice were inoculated with 3,000,000 tumor cells, and then when the tumor volume reached 63-124mm³(average TV range of 81-89mm per group³) At the time, 10 mice per group were randomly grouped. Animals were dosed on and after day 0 as follows: every three days 100ul PBS (vehicle) was injected subcutaneously (group 1); every three days near the tumor [ around the tumor ]]0.1 mg/mouse IMM-101 subcutaneously (group 2); twice weekly anti-PD 1 intraperitoneal [ RMP1-14]Administration (group 3), or twice weekly anti-PD-1 [ RMP1-14 ]]And IMM-101 every 3 days (group 4;intraperitoneal and peritumoral, respectively). Mice were dosed until 3000mm due to moribund or maximal Tumor Volume (TV)³(for later).

The results are shown in FIGS. 15-19, showing mean TV +/-SE (FIG. 15), mean TV without SE (FIG. 16), mean TV without SE up to study day 37 (FIG. 17), median TV (FIG. 18), and median TV up to study day 37 (FIG. 19).

Calculation of tumor growth inhibition (%) for each group and study day indicated that group 3 (anti-PD 1 only) exhibited a maximum% TGI of-9.24% at study day 30 (when all mice in this group were terminated), while group 4 (peri-oncotic IMM-101 plus anti-PD-1) exhibited a% TGI of 56.22% at study day 41 and group 2 (peri-oncotic IMM-101 only) exhibited a% TGI of 46.93% at study day 41.

As can be seen from figures 15-19, the combined efficacy of IMM-101 and anti-PD 1 was significantly improved in this checkpoint refractory mouse model compared to anti-PD 1 alone, in terms of% TGI values, particularly when IMM-101 alone or in combination with anti-D1 was administered subcutaneously near the tumor. Furthermore, the combination or IMM-101 monotherapy also resulted in greater survival rates than anti-PD 1 alone.