Hematopoietic stem cells in combination therapy with immune checkpoint inhibitors for cancer

Abstract

Combination treatments are provided that result in synergistic effects in the treatment of a disease selected from cancer or infectious diseases. [Solution] A method for treating a disease selected from cancer or infectious disease, comprising administering to a subject having the disease one or more immune checkpoint inhibitors in an amount effective to treat the disease, and administering hematopoietic stem cells to the subject, preferably further comprising administering to the subject a hematopoietic stem cell mobilizing agent.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 62 / 199,916 filed on 31 July 2015, U.S. Provisional Application No. 62 / 296,826 filed on 18 February 2016, U.S. Provisional Application No. 62 / 296,849 filed on 18 February 2016, and U.S. Provisional Application No. 62 / 296,866 filed on 18 February 2016, all of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of Disclosure The use of immune checkpoint inhibitors, which bind to immune checkpoint molecules such as programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), cytotoxic T lymphocyte-associated antigen (CTLA-4), or V-domain Ig suppressor of T cell activation (VISTA) to cause immune checkpoint blockade, is a promising approach being studied for the treatment of cancer and infectious diseases. Despite impressive therapeutic responses in clinical trials of many cancers, not all subjects with these cancers respond to immune checkpoint blockade. Furthermore, there are many cancers in which the therapeutic response to treatment with antibodies that bind to immune checkpoint molecules (e.g., PD-1 or CTLA-4) is not evident.

[0003] Enhancing CD4 and CD8 T cell activity against various cell types, including cancer cells, is another approach being studied to treat cancer and infections. One strategy involves stimulating T lymphocytes with an antigen, amplifying them ex vivo, and then injecting them into the target. This is a form of adoptive cell therapy (ACT). Certain ACT strategies have been shown in early clinical trials to induce cancer regression. ACT may be particularly useful in treating cancer and / or infections that arise after immunodeprivation and hematopoietic stem cell transplantation (HSCT). Another approach being studied to treat cancer is hematopoietic stem cell transplantation (HSCT) and / or hematopoietic stem cell (HSC) recruitment. HSCT and / or HSC recruitment may enhance the effects of certain cell-based immunotherapies when combined with treatments that induce mild lymphopenia.

[0004] Immunotherapy alone, such as anti-PD1, anti-PD-L1, anti-CTLA-4, or anti-VISTA-mediated blockade, and administration of HSCs or HSC mobilizers alone, have not shown clinical efficacy in many subjects with different cancers. However, according to this disclosure, the combination of HSC transplantation and immune checkpoint blockade (e.g., anti-PD-1-mediated blockade or anti-VISTA-mediated blockade) has been found to be synergistic in the treatment of cancer. This synergy enables the treatment of cancers resistant to immune checkpoint blockade by treatment with immune checkpoint inhibitors and HSC transplantation, and can lead to significant long-term regression of checkpoint inhibitor-resistant cancers. The findings of this disclosure that combined treatment with anti-PD-1 antibodies and hematopoietic stem cell transplantation reverses resistance to immune checkpoint blockade using anti-PD-1 antibody monotherapy have been demonstrated in multiple brain tumor models (e.g., brainstem glioma, corticoblastoma, and medulloblastoma). [Overview of the project]

[0005] The inventors have made a unique observation that bone marrow-derived hematopoietic stem cells (HSCs) administered to experimental tumor-carrying mice promote the persistence and survival of activated interferon-gamma (IFNγ) secreting T cells in the tumor microenvironment and tumor inflow lymph nodes in mice that have undergone immune checkpoint blockade with immune checkpoint inhibitors. Immune checkpoint blockade using anti-PD-1, anti-PD-L1, or anti-CTLA-4 monoclonal antibodies has been shown to be an important and effective modality in many cancers (such as melanoma and non-small cell lung cancer), and is currently being evaluated for its efficacy against many human tumors and its potential as a tumor predictive biomarker (Mahoney et al., 2015; Shih et al., 2014; Dolan et al., 2014, these are incorporated herein by reference in their entirety).

[0006] Immune checkpoint blockade using anti-VISTA monoclonal antibodies is evaluated in mouse tumor models by CD8 expression, IFN-γ, and TNF-α. + It has been shown to increase T cell activation but not to affect tumor growth (Kondo et al. 2015. J. of Immun. V194). VISTA induces immunosuppressive activity on T cells both in vitro and in vivo, and VISTA blockade enhances T cell-mediated immunity in autoimmune disease models (Wang et al. 2011. JEM 208(3):577-92). VISTA blockade in combination with other immunotherapies, such as HSCT or HSC mobilization, may be an important mediator in controlling the development of autoimmunity and the immune response to cancer.

[0007] Despite impressive clinical responses in many cancers, responses to treatment with immune checkpoint inhibitors (e.g., anti-immune checkpoint antibodies) are currently observed only in a subset of treated patients. The results disclosed herein demonstrate that combined treatment of HSC transfer with immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, anti-CTLA-4, or anti-VISTA antibodies, is synergistic in treating tumors resistant to anti-immune checkpoint antibodies alone, where the combination is curative in a significant portion of the treated animals. The combination of HSCT and immune checkpoint inhibitor treatment is highly effective, whereas HSCT alone or immune checkpoint inhibitor treatment alone does not yield immunological or clinical effects. These results demonstrate a previously undescribed, potent synergistic effect of HSC transfer with immune checkpoint inhibitor treatment for treating cancer. While we do not wish to be bound by any theory in this disclosure, we offer insight into the mechanism by demonstrating that this combination leads to a sustained increase in IFNγ-positive T cells within the tumor microenvironment. The inventors propose that a novel synergistic combination of immune checkpoint inhibitors and HSC transfer (and / or HSC recruitment) has a significant impact on antitumor immunity. Hereinafter, it is proposed that administration of HSCs, in combination with one or more immune checkpoint inhibitors, to tumor-carrying hosts may lead to increased IFNγ secretion. Such treatment may be particularly useful in hosts with subcutaneous, systemic, or intracranial tumors, as well as in tumor-carrying hosts undergoing radiation or chemotherapy, and in hosts not undergoing radiation or chemotherapy. This disclosure supports the synergistic effects of combined treatment using immune checkpoint inhibitors and HSCs and / or HSC recruiters.

[0008] According to one aspect of the present disclosure, a method is provided for treating a disease selected from cancer or infectious diseases, comprising administering one or more immune checkpoint inhibitors to a subject having the disease in an effective amount for treating the disease, and administering hematopoietic stem cells to the subject. One aspect of this disclosure provides a method for treating a disease selected from cancer or infectious diseases, comprising administering to a subject having the disease one or more immune checkpoint inhibitors in an effective amount for treating the disease, and administering a hematopoietic stem cell mobilizer.

[0009] One aspect of this disclosure provides a method for treating a disease selected from cancer or an infection in a subject receiving immune checkpoint inhibitor therapy for the disease, the method comprising administering to the subject hematopoietic stem cells in an effective amount for treating the disease in combination with the immune checkpoint inhibitor therapy. One aspect of this disclosure provides a method for treating a disease selected from cancer or an infection in a subject receiving hematopoietic stem cell transplantation therapy for the disease, the method comprising administering to the subject one or more immune checkpoint inhibitors in an effective amount for treating the disease in combination with hematopoietic stem cell transplantation therapy.

[0010] In the aspects described above and in any of the following embodiments, the disease may be resistant, for example, to monotherapy with one or more immune checkpoint inhibitors. In the aspects described above and in any of the following embodiments, one or more immune checkpoint inhibitors are, for example, antagonists of: programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), V-domain Ig suppressor of T cell activation (VISTA), programmed death ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase, B7 family inhibitory ligand B7-H3, B7 family inhibitory ligand B7-H4, lymphocyte activation gene 3 (LAG3), 2B4, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR), killer inhibitory receptor, and / or signaling and transcriptional activator (STAT) 3. In a particular embodiment, one or more immune checkpoint inhibitors are each an antagonist of programmed death 1 (PD-1), an antagonist of programmed death ligand 1 (PD-L1), an antagonist of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and / or an antagonist of V-domain Ig suppressor of T cell activation (VISTA).

[0011] In any of the aspects and embodiments described above, the PD-1 antagonist is, for example, a drug that binds to and antagonizes PD-1. Such a drug may be, for example, a peptide that binds to PD-1. Such a drug may be a humanized antibody that selectively binds to PD-1. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, pizilizumab, MEDI-0680, REGN2810, or AMP-224. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, or pizilizumab. In some embodiments, the antagonist is (i) an antisense molecule against PD-1, (ii) adnectin against PD-1, (iii) a single-stranded or double-stranded RNAi inhibitor of PD-1, and / or (iv) a small molecule inhibitor of PD-1.

[0012] In any of the aspects and embodiments described above, the PD-L1 antagonist is, for example, a drug that binds to and antagonizes PD-L1. Such a drug may be, for example, a peptide that binds to PD-L1. Such a drug may be a humanized antibody that selectively binds to PD-L1. In some embodiments, humanized antibodies that selectively bind to PD-L1 are BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab. In some embodiments, the antagonist is (i) an antisense molecule against PD-L1, (ii) adnectin against PD-L1, (iii) a single-stranded or double-stranded RNAi inhibitor of PD-L1, or (iv) a small molecule inhibitor of PD-L1.

[0013] In any of the aspects and embodiments described above, the CTLA-4 antagonist is, for example, a drug that binds to and antagonizes CTLA-4. Such a drug may be, for example, a peptide that binds to CTLA-4. Such a drug may be a humanized antibody that selectively binds to CTLA-4. In some embodiments, the humanized antibody that selectively binds to CTLA-4 is ipilimumab or tremelimumab. In some embodiments, the CTLA-4 antagonist is (i) an antisense molecule against CD80, CD86 and / or CTLA-4, (ii) adnectin against CD80, CD86 and / or CTLA-4, (iii) a single-stranded or double-stranded RNAi inhibitor of CD80, CD86 and / or CTLA-4, or (iv) a small molecule inhibitor of CD80, CD86, or CTLA-4.

[0014] In any of the aspects and embodiments described above, the VISTA antagonist is, for example, a drug that binds to and antagonizes VISTA. Such a drug may be, for example, a peptide. Such a drug may be an inhibitory antibody against VISTA. In some embodiments, the drug that binds to and antagonizes VISTA is a humanized antibody. In some embodiments, the drug that binds to and antagonizes VISTA is (i) an antisense molecule against VISTA, (ii) adnectin against VISTA, (iii) a single-stranded or double-stranded RNAi inhibitor of VISTA, or (iv) a small molecule inhibitor of VISTA.

[0015] In any of the embodiments described above, the immune checkpoint inhibitor is administered on a different day from the hematopoietic stem cell transplant or hematopoietic stem cell mobilization agent. In any of the embodiments described above, the immune checkpoint inhibitor is administered on the same day as the hematopoietic stem cell transplant or hematopoietic stem cell mobilization agent. In any of the embodiments described above, the immune checkpoint inhibitor is administered on a different day from the hematopoietic stem cell transplant or hematopoietic stem cell mobilization agent, but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months of the hematopoietic stem cell transplant or mobilization agent. In any of the foregoing aspects, the immune checkpoint inhibitor is administered, for example, intravenously or subcutaneously.

[0016] In any of the foregoing aspects, the method further comprises administering a hematopoietic stem cell mobilizing agent to the subject. In an exemplary aspect, the mobilizing agent is granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), pegylated G-CSF (pegfilgrastim), lenograstim, a glycosylated form of G-CSF, C-X-C motif chemokine 2 (CXCL2), C-X-C chemokine receptor type 4 (CXCR-4), or plerixafor. In any of the foregoing aspects, the disease is, for example, cancer, and the cancer is melanoma, squamous cell carcinoma, basal cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular tumor, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain tumor, glioblastoma, medulloblastoma, epithelioma, angiosarcoma, angioendothelioma, mastocytoma, primary liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic neoplasm formation, lymphoma, mesothelioma, glioblastoma, low-grade glioma, high-grade glioma, pediatric brain tumor, medulloblastoma, or metastatic cancer thereof.

[0017] In any of the foregoing aspects, the cancer is a metastatic or refractory cancer of the brain, lung, breast, or melanoma. In any of the foregoing aspects, the cancer is a metastatic brain tumor derived from non-small cell lung cancer, a metastatic brain tumor derived from melanoma, or a metastatic brain tumor derived from breast cancer. In any of the foregoing aspects, the cancer is glioblastoma, low-grade glioma, high-grade glioma, pediatric brain tumor, or medulloblastoma. In any of the foregoing aspects, the disease is, for example, an infectious disease. In any of the foregoing aspects, the infectious disease is a chronic infectious disease. In any of the foregoing aspects, the infectious disease is any hepatitis, adenovirus, polyomavirus such as BK, human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and / or C, vesicular stomatitis virus (VSV), staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), streptococcal species including Streptococcus pneumoniae, or a post-transplant infectious disease. In any of the foregoing aspects, the infectious disease is hepatitis A, hepatitis B, or hepatitis C.

[0018] In any of the foregoing aspects, the source of hematopoietic stem cells is, for example, bone marrow, lineage-depleted cells (lin-), cKit+ purified lineage-negative bone marrow-derived cells, Sca+ purified lineage-negative bone marrow-derived cells, cKit+Sca+ purified bone marrow-derived cells, mobilized from host bone marrow using GM-CSF, G-CSF, AMD3100, plerixafor, or molecule 1,1’-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane], umbilical cord blood or umbilical cord blood-derived stem cells, human leukocyte antigen (HLA)-matched blood, mesenchymal stem cells derived from blood or bone marrow, hematopoietic stem cells differentiated from induced pluripotent stem cells, mobilized peripheral blood, peripheral blood, a subset of hematopoietic stem cells including lin- cells purified by the CCR2+ marker, lineage-negative purified peripheral blood, or CD34+-enriched peripheral blood. In any of the foregoing aspects, the source of hematopoietic stem cells is bone marrow, peripheral blood, umbilical cord blood, or induced pluripotent stem cells. In any of the foregoing aspects, the source of hematopoietic stem cells is autologous. In some aspects, the source of hematopoietic stem cells is allogeneic and the donor cells are HLA-matched to the recipient.

[0019] In any of the embodiments described above, a sample containing hematopoietic stem cells is obtained and optionally treated in vitro to increase the number of stem cells in the sample before administering the hematopoietic stem cells to a subject. In any of the embodiments described above, a sample containing hematopoietic stem cells is obtained and optionally treated in vitro to increase the percentage of stem cells in the sample before administering the hematopoietic stem cells to a subject. The sample may be autologous or not. In any of the embodiments described above, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percent of the hematopoietic stem cells are CCR2-positive (CCR2+), CD34-positive (CCR2+), and / or lineage-negative (lin-) cells. In any of the embodiments described above, 20% to 98% of the hematopoietic stem cells for administration to the subject, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, are CCR2-positive (CCR2+), CD34-positive (CD34+), or lineage-negative (lin-).

[0020] In any of the embodiments described above, hematopoietic stem cells for administration to a subject are optionally enriched ex vivo with CCR2-positive (CCR2+) cells, CD34-positive (CD34+) cells, and / or lineage-negative (lin-) cells before administration to the subject. In any of the embodiments described above, hematopoietic stem cells are optionally treated ex vivo to remove CCR2-negative (CCR2-) cells before administration to the subject. In any of the embodiments described above, hematopoietic stem cells are selected for CCR2+, CD34+, and / or lin- cells before administration to the subject by flow cytometry analysis, microbead-based isolation, adhesion assay, and / or ligand-based selection. In some embodiments, cells are selected by ligand-based selection, where the ligand is a CCR2 ligand known as CCL2.

[0021] In any of the aforementioned embodiments, the effect of the treatment on the disease is evaluated, for example, by measuring the secretion of interferon-gamma (IFNγ) by T cells obtained from the target tumor microenvironment or tumor inflow area lymph nodes, and a synergistic effect is observed when the presence of IFNγ increases with combination therapy. In any of the embodiments described above, adoptive cell therapy (ACT) may also be administered to the subject. In any of the embodiments described above, adoptive cell therapy (ACT) is administered to the subject at a time sufficiently close to that of at least one of the aforementioned treatments to enhance the treatment of the disease.

[0022] In any of the embodiments described above, chemotherapy or radiation may be administered to the subject. In any of the embodiments described above, chemotherapy or radiation may be administered to the subject at a sufficiently close time to at least one of the treatments to enhance the treatment of the disease. In such embodiments, hematopoietic stem cells may be administered to the subject after the completion of the radiation treatment. In such embodiments, hematopoietic stem cells may be administered to the subject after the completion of the chemotherapy treatment. In such embodiments, hematopoietic stem cells may be administered to the subject within six weeks after the completion of the chemotherapy or radiation treatment. In such embodiments, hematopoietic stem cells may be administered to the subject 0, 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 6 weeks after the completion of the chemotherapy or radiation treatment. In such embodiments, immune checkpoint inhibitors may be administered before, concurrently with, or after the radiation or chemotherapy treatment. In such embodiments, immune checkpoint inhibitors may be administered to the subject 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the completion of the chemotherapy or radiation treatment.

[0023] In any of the embodiments described above, hematopoietic stem cells may be treated with one or more different cytokines before administration to the hematopoietic stem cells. In any of the embodiments described above, hematopoietic stem cells may be administered to the subject simultaneously with one or more different cytokines. In any of the embodiments described above, treatment of HSCs with one or more different cytokines can further enhance the effect of combined immune checkpoint inhibitor therapy and hematopoietic stem cell transplantation therapy. In any of the embodiments described above, administration of HSCs to the subject simultaneously with the administration of one or more different cytokines can further enhance the effect of combined immune checkpoint inhibitor therapy and hematopoietic stem cell transplantation therapy. In some embodiments, the one or more different cytokines are IFNγ, TNFα, GM-CSF, G-CSF, Fl3-ligand, IL-1β, IL-4 and / or IL-6. In any of the embodiments described above, hematopoietic stem cells are treated with one or more cytokines on the same day, one day, two days, three days, four days, or five days prior to the administration of HSCs to the subject. In any of the embodiments described above, hematopoietic stem cells are treated with one or more cytokines one, two, three, four, or five days before being administered to the HSCs.

[0024] According to one aspect of this disclosure, hematopoietic stem cells are provided for use in treating subjects having cancer or an infection, wherein the subjects have undergone concurrent immune checkpoint inhibitor treatment with one or more immune checkpoint inhibitors. According to one aspect of this disclosure, hematopoietic stem cells enriched with CCR2+, CD34+, and / or lin- cells are provided for use in the treatment of subjects being treated for a disease with one or more immune checkpoint inhibitors.

[0025] According to one aspect of this disclosure, hematopoietic stem cells from which CCR2- cells have been substantially removed are provided for use in the treatment of subjects who are being treated for a disease with one or more immune checkpoint inhibitors. According to one aspect of this disclosure, an immune checkpoint inhibitor is provided for use in treating a subject having cancer or an infectious disease, wherein the subject is concurrently treated with hematopoietic stem cells.

[0026] According to one aspect of this disclosure, an immune checkpoint inhibitor is provided for use in treating a subject having cancer or an infectious disease, wherein the subject is undergoing concurrent treatment with hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilizer. In any of the aforementioned aspects, the disease may be resistant to monotherapy with one or more immune checkpoint inhibitors. In any of the aforementioned aspects, more than one different immune checkpoint inhibitors may be used simultaneously in combination with hematopoietic stem cells and / or hematopoietic stem cell mobilizers for the treatment of subjects with cancer or infection.

[0027] In any of the aforementioned aspects, immune checkpoint inhibitors are antagonists of, for example, the following: programmed death ligand 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), V-domain Ig suppressor of T cell activation (VISTA), programmed death ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase, B7 family inhibitory ligand B7-H3, B7 family inhibitory ligand B7-H4, lymphocyte activation gene 3 (LAG3), 2B4, B and T lymphocyte attenuator (BTLA), T cell membrane protein 3 (TIM3), adenosine A2a receptor (A2aR), and / or killer inhibitory receptors. In any of the aforementioned aspects, immune checkpoint inhibitors are antagonists of: programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and / or V-domain Ig suppressor of T cell activation (VISTA).

[0028] In some embodiments, immune checkpoint inhibitors are programmed death-1 (PD-1) antagonists. In some embodiments, immune checkpoint inhibitors are programmed death-ligand-1 (PD-L1) antagonists. In some embodiments, immune checkpoint inhibitors are cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antagonists. In some embodiments, immune checkpoint inhibitors are T-cell activation V-domain Ig suppressor (VISTA) antagonists. A PD-1 antagonist can be, for example, a drug that binds to and antagonizes PD-1. In some embodiments, the drug that binds to and antagonizes PD-1 is a peptide that binds to PD-1. In some embodiments, the drug that binds to and antagonizes PD-1 is a humanized antibody that selectively binds to PD-1. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, pizilizumab, MEDI-0680, REGN2810, or AMP-224. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, or pizilizumab.

[0029] A PD-L1 antagonist can be, for example, a drug that binds to and antagonizes PD-L1. In some embodiments, the drug that binds to and antagonizes PD-L1 is a peptide that binds to PD-L1. In some embodiments, the drug that binds to and antagonizes PD-L1 is a humanized antibody that selectively binds to PD-L1. In some embodiments, the humanized antibody that selectively binds to PD-L1 is BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab.

[0030] A CTLA-4 antagonist may be, for example, a drug that binds to and antagonizes CTLA-4. In some embodiments, the drug that binds to and antagonizes CTLA-4 is a peptide that binds to CTLA-4. In some embodiments, the drug that binds to and antagonizes CTLA-4 is a humanized antibody that selectively binds to CTLA-4. In some embodiments, the humanized antibody that selectively binds to a CTLA-4 inhibitor is ipilimumab or tremelimumab. In some embodiments, a CTLA-4 antagonist is (i) an antisense molecule against CD80, CD86, and / or CTLA-4, (ii) adnectin against CD80, CD86, and / or CTLA-4, (iii) a single-stranded or double-stranded RNAi inhibitor of CD80, CD86, and / or CTLA-4, or (iv) a small molecule inhibitor of CD80, CD86, or CTLA-4.

[0031] A VISTA antagonist can be, for example, a drug that binds to and antagonizes VISTA. In some embodiments, the drug that binds to and antagonizes VISTA is a peptide. In some embodiments, the drug that binds to and antagonizes VISTA is an inhibitory antibody against VISTA. In some embodiments, the drug that binds to and antagonizes VISTA is a humanized antibody. In some embodiments, the drug that binds to and antagonizes VISTA is (i) an antisense molecule against VISTA, (ii) adnectin against VISTA, (iii) a single-stranded or double-stranded RNAi inhibitor of VISTA, or (iv) a small molecule inhibitor of VISTA.

[0032] In some embodiments, immune checkpoint inhibitors are administered on a different day than hematopoietic stem cell transplantation or hematopoietic stem cell mobilization. In some embodiments, immune checkpoint inhibitors are administered on the same day as hematopoietic stem cell transplantation or hematopoietic stem cell mobilization. In some embodiments, immune checkpoint inhibitors are administered on a different day than hematopoietic stem cell transplantation or hematopoietic stem cell mobilization, but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months prior to hematopoietic stem cell transplantation or hematopoietic stem cell mobilization. In some embodiments, immune checkpoint inhibitors are administered intravenously or subcutaneously.

[0033] In any of the embodiments described above, the method optionally further includes administering a hematopoietic stem cell mobilizer to the target. In some embodiments, the mobilizer is granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), pegylated G-CSF (pegylated G-CSF), lenoglatism, glycosylated forms of G-CSF, CXC motif chemokine 2 (CXCL2), CXC chemokine receptor type 4 (CXCR-4), or prelixafor.

[0034] In any of the embodiments described above, disease is, for example, cancer, and cancer is melanoma, squamous cell carcinoma, basal cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular tumor, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain tumor, glioblastoma, medulloblastoma, ependymal cell tumor, angiosarcoma, hemangioendothelioma, mast cell tumor, primary liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic neoplasm, lymphoma, mesothelioma, glioblastoma, low-grade glioma, high-grade glioma, pediatric brain tumor, medulloblastoma, or metastatic cancers thereof. In some embodiments, cancer is metastatic or refractory cancer of the brain, lung, breast, or melanoma. In some embodiments, cancer is metastatic brain tumor originating from non-small cell lung cancer, metastatic brain tumor originating from melanoma, or metastatic brain tumor originating from breast cancer. In some aspects, cancer is glioblastoma, low-grade glioma, high-grade glioma, pediatric brain tumor, or medulloblastoma.

[0035] In some embodiments, the disease is, for example, an infection. In some embodiments, the infection is a chronic infection. In any of the embodiments described above, the infection is any hepatitis, adenovirus, polyomavirus such as BK, human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and / or C, varicella stomatitis virus (VSV), staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), streptococcal species including Streptococcus pneumoniae, or a post-transplant infection. In some embodiments, the infection is hepatitis A, hepatitis B, or hepatitis C.

[0036] In any of the embodiments described above, the source of hematopoietic stem cells is, for example, bone marrow, myeloid-cleared cells (lin-), cKit+ purified lineage-negative bone marrow-derived cells, Sca+ purified lineage-negative bone marrow-derived cells, cKit+Sca+ purified bone marrow-derived cells, mobilization from host bone marrow using GM-CSF, G-CSF, AMD3100, prelixafor, or mobilization from host bone marrow using the molecule 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane], umbilical cord blood or umbilical cord blood-derived stem cells, human leukocyte antigen (HLA)-matched blood, mesenchymal stem cells derived from blood or bone marrow, hematopoietic stem cells differentiated from induced pluripotent stem cells, mobilized peripheral blood, peripheral blood, a hematopoietic stem cell subset including lin- cells purified with a CCR2+ marker, lineage-negative purified peripheral blood, or CD34+ enriched peripheral blood. In some embodiments, the source of hematopoietic stem cells is bone marrow, peripheral blood, umbilical cord blood, or induced pluripotent stem cells.

[0037] In any of the aforementioned embodiments, the source of hematopoietic stem cells is autologous. In any of the aforementioned embodiments, the hematopoietic stem cell source is allogeneic, and the donor cells are HLA-compatible with the recipient. In some embodiments, a sample containing hematopoietic stem cells is obtained, which is treated in vitro to increase the percentage of stem cells in the sample before administering hematopoietic stem cells to a subject. In some embodiments, a sample containing hematopoietic stem cells is obtained, which is treated in vitro to increase the percentage of stem cells in the sample before administering hematopoietic stem cells to a subject. In some embodiments, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percent of the hematopoietic stem cells are CCR2-positive (CCR2+), CD34-positive (CCR2+), and / or lineage-negative (lin-) cells. In some embodiments, between 20% and 98% of hematopoietic stem cells for administration to a subject, e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, are CCR2-positive (CCR2+), CD34-positive (CD34+), or lineage-negative (lin-). In some embodiments, hematopoietic stem cells for administration to a subject are enriched ex vivo with CCR2-positive (CCR2+) cells, CD34-positive (CD34+) cells, and / or lineage-negative (lin-) cells before administration to the subject. In some embodiments, hematopoietic stem cells are treated ex vivo before administration to the subject to remove CCR2-negative (CCR2-) cells. In some embodiments, hematopoietic stem cells are selected for CCR2+, CD34+, and / or lin- cells prior to administration to the subject by flow cytometry analysis, microbead-based isolation, adhesion assay, and / or ligand-based selection. In some embodiments, cells are selected by ligand-based selection, where the ligand is the CCR2 ligand known as CCL2.

[0038] In some embodiments, the effectiveness of a treatment for a disease can be evaluated by measuring interferon-gamma (IFNγ) secretion by T cells obtained from the target tumor microenvironment or tumor inflow lymph nodes, where a synergistic effect is observed when the presence of IFNγ increases with combination therapy. In any of the embodiments described above, adoptive cell therapy (ACT) may be administered to the subject at will. In some embodiments, adoptive cell therapy (ACT) is administered to the subject at a time sufficiently close to at least one of the aforementioned treatments to enhance the treatment of the disease.

[0039] In any of the embodiments described above, chemotherapy or radiation may be administered to the subject. In some embodiments, chemotherapy or radiation is administered to the subject at a time sufficiently close to that of at least one of the treatments to enhance the treatment of the disease. In any of the embodiments described above, hematopoietic stem cells can be treated with one or more different cytokines before being administered to the hematopoietic stem cells. In some embodiments, treatment of HSCs with one or more different cytokines can further enhance the efficacy of combination therapy with immune checkpoint inhibitor therapy and hematopoietic stem cell transplantation therapy. In some embodiments, the one or more different cytokines are IFNγ, TNFα, IL-1β and / or IL-6.

[0040] One aspect of the present disclosure provides a method for treating a subject, comprising: administering a stem cell mobilizer to the subject; collecting hematopoietic stem cells from the subject; enriching the collected stem cells with CCR2-positive (CCR2+), CD34-positive (CD34+), or lineage-negative (lin-) cells; optionally removing the collected stem cells or CCR2- cells; administering the enriched collected stem cells to the subject; and administering an immune checkpoint inhibitor to the subject. [Brief explanation of the drawing]

[0041] [Figure 1A] Figures 1A, 1B, and 1C show experimental results from tumor-bearing mice that underwent adoptive transfer of tumor-reactive T cells with or without co-transfer of high-strength stem cells (HSCs). Lymph nodes in the tumor inflow area were dissected in both groups and analyzed for T cell activation. Figure 1A shows a real-time PCR array demonstrating that mice that received co-transfer of HSCs had increased IFNγ compared to mice that did not. [Figure 1B] Figure 1B shows a flow cytometry analysis of IFNγ secretion by adopted tumor-specific T cells. [Figure 1C] Figure 1C shows the quantification of the results from Figure 1B using flow cytometry, confirming the observations in Figure 1B.

[0042] [Figure 2A] Figure 2A shows the expected results when 10,000 astrocytoma cells were transplanted intracranially into YETI mice (IFNγ reporter mice whose cells fluoresce when IFNγ is produced). On day 3, the mice received intravenous injection of HSCs, intraperitoneal injection of anti-PD-1 antibody, or both. It was easy to determine whether T cell activation was occurring using YETI host mice. After 30 days, the tumors were excised and sectioned into 500 mm slices using a tissue slicer. [Figure 2B] Figure 2B shows that fluorescence was detected using an Olympus IX70 inverted fluorescence microscope and quantified by determining the MFI per slice. The group that received anti-PD-1 antibody and HSC had significantly higher YFP expression than the other groups. [Figure 2C] Figure 2C shows the results of a subsequent study conducted to determine whether the combination of HSCs and anti-PD-1 antibodies demonstrated superior antitumor protection. 10,000 astrocytoma cells were intracranially administered to C57BL / 6 mice. On day 3, the mice were given intravenous injection of HSCs, intraperitoneal injection of anti-PD-1 antibodies, or both. The groups followed humane endpoints. 40% of the mice treated with anti-PD-1 and HSCs showed complete remission.

[0043] [Figure 3] Figure 3 shows that a specific HSC subset mediates enhanced antitumor immunity against malignant gliomas. [Figure 4] Figure 4 shows that a specific HSC subset mediates enhanced antitumor immunity against medulloblastoma.

[0044] [Figure 5] Figure 5 shows the survival of immunocompetent C57BL / 6 mice treated with intracranial tumors, either untreated or treated with HSCs, anti-PD1 antibody, HSCs and anti-PD1 antibody, irradiation and HSCs, irradiation and anti-PD1 antibody, or irradiation, HSCs, and anti-PD1 antibody. Anti-PD1 antibody is one example of the treatment. [Figure 6] Figure 6 shows the survival of immune-responsive C57BL / 6 mice with intracranial tumors, either untreated or treated with HSCs, anti-VISTA antibody, or HSCs and anti-PD1 antibody.

[0045] [Figure 7] Figure 7 shows the survival of immune-responsive C57BL / 6 mice with intracranial tumors, treated with untreated or lineage-negative hematopoietic stem cells (Lin-HSC), CCR2-nonexpressing HSCs (CCR2neg HSC), CCR2-expressing HSCs (CCR2pos HSC), αPD1, αPD1+HSC, αPD1+CCR2neg HSC, or αPD1+CCR2pos HSC.

[0046] [Figure 8A-1] Figures 8A and 8B show the percentage of interferon-gamma (IFNγ) secreting cells among total CD3+ T cells in the tumor microenvironment (Figure 8A), and the quantification of flow cytometry analysis (Figure 8B), determined by flow cytometry analysis of yellow fluorescent protein (YFP) / IFNγ+ / CD3+ lymphocytes in the tumor microenvironment in untreated mice and mice treated with HSC, anti-PD1, or both HSC and anti-PD1. [Figure 8A-2] Figures 8A and 8B show the percentage of interferon-gamma (IFNγ) secreting cells among total CD3+ T cells in the tumor microenvironment (Figure 8A), and the quantification of flow cytometry analysis (Figure 8B), determined by flow cytometry analysis of yellow fluorescent protein (YFP) / IFNγ+ / CD3+ lymphocytes in the tumor microenvironment in untreated mice and mice treated with HSC, anti-PD1, or both HSC and anti-PD1. [Figure 8B] Figures 8A and 8B show the percentage of interferon-gamma (IFNγ) secreting cells among total CD3+ T cells in the tumor microenvironment (Figure 8A), and the quantification of flow cytometry analysis (Figure 8B), determined by flow cytometry analysis of yellow fluorescent protein (YFP) / IFNγ+ / CD3+ lymphocytes in the tumor microenvironment in untreated mice and mice treated with HSC, anti-PD1, or both HSC and anti-PD1.

[0047] [Figure 9] Figure 9 shows the expression of 92 genes involved in the T cell activation / inflammatory pathway in immune-responsive C57BL / 6 mice with intracranial tumors, either untreated or treated with HSC, αPD-1, or HSC and αPD-1.

[0048] Detailed explanation The following detailed descriptions are provided for the purpose of illustrating specific aspects of this disclosure. It should be understood that other aspects may be contemplated and made without departing from the scope or spirit of this disclosure. Therefore, the following detailed descriptions should not be construed as restrictive. Scientific and technical terms used herein have their meanings as commonly used in the art unless otherwise specified. The definitions provided herein are for the purpose of facilitating the understanding of certain terms that are frequently used herein and do not limit the scope of this disclosure. The singular forms “a,” “an,” and “the” encompass the plural unless the context explicitly indicates otherwise. The term “or” is used generally to include “and / or” unless the context explicitly indicates otherwise.

[0049] Cancer. The therapies described herein include treatment of existing or established cancers, i.e., cancers present and detectable in the subject. Furthermore, treatment of precancerous lesions (i.e., adenomatous polyps or cytodysplasia) to prevent the development of cancer is also envisioned. Cancers treatable in accordance with this disclosure include: melanoma, squamous cell carcinoma, basal cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular tumor, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain tumor, glioblastoma, medulloblastoma, ependymal cell tumor, angiosarcoma, hemangioendothelioma, mast cell tumor, primary liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, gastrointestinal cancer, renal cell carcinoma, hematopoietic neoplasm, lymphoma, mesothelioma, or metastatic cancers thereof. In aspects of this disclosure, cancers addressed in this disclosure include glioblastoma, low-grade glioma, high-grade glioma, brainstem glioma, cortical glioblastoma, pediatric brain tumors, and medulloblastoma. In aspects of this disclosure, cancer is invasive intracranial glioma. In aspects of this disclosure, cancer is metastatic or refractory cancer of the brain, lung, breast, or melanoma. In aspects of this disclosure, cancer is metastatic brain tumor originating from non-small cell lung cancer, metastatic brain tumor originating from melanoma, or metastatic brain tumor originating from breast cancer. In aspects of this disclosure, cancer is brainstem glioma, cortical glioblastoma, and medulloblastoma.

[0050] Infectious Diseases. This disclosure is also useful in relation to the treatment of infectious diseases. Generally, opportunistic pathogenic microorganisms can be classified into viruses, fungi, parasites, and bacteria. Exemplary pathogenic viral microorganisms that cause disease in humans include (but are not limited to) filoviruses, herpesviruses, hepatitis viruses, retroviruses, human immunodeficiency virus (HIV), orthomyxoviruses, paramyxoviruses, togaviruses, picornaviruses, papovaviruses, and gastroenteritis viruses. Examples of pathogenic bacteria that cause serious human diseases include Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and E. faecium, Streptococcus pneumoniae, and Gram-negative bacteria: Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonas maltophila, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, and Salmonella species. Examples of pathogenic protists that cause human diseases include (but are not limited to) malaria (e.g., Plasmodium falciparum and M. ovale), trypanosomiasis (sleeping sickness) (e.g., Trypanosoma cruzei), leishmaniasis (e.g., Leischmania donovani), and amoebiasis (e.g., Entamoeba histolytica). Exemplary pathogenic fungi that cause or are associated with human disease include, but are not limited to, Candida albicans, Histoplasma neoformans, Coccidioides immitis, and Penicillium marneffei. In some embodiments, infectious organisms are those involved in chronic infections.Particularly important diseases include hepatitis, adenoviruses, polyomaviruses such as BK, human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, varicella stomatitis virus (VSV), staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), streptococcal species including Streptococcus pneumoniae, and post-transplant infections.

[0051] Antibodies, or immunoglobulins, are glycoproteins containing two identical light chains (L chains), each containing approximately 200 amino acids, and two identical heavy chains (H chains), generally at least twice as long as the L chains. The paratopes of antibodies are specific to a particular epitope of an antigen, and their spatial complementarity (binding) either "labels" the microorganism for further action or directly neutralizes its action. Antibodies communicate with other components of the immune response through their crystalline fragment (Fc) region, which contains conserved glycosylation sites. Five Fc regions exist, resulting in five distinct antibody isotypes: IgA, IgD, IgE, IgG, and IgM. IgD functions as an antigen receptor on B cells not exposed to the antigen, activating basophils and mast cells, leading to the production of antimicrobial factors. IgG is expressed in four forms and provides the majority of antibody-based immunity against invading pathogens. IgM is expressed as a monomer on the surface of B cells and as a pentamer in secretory form. IgM eliminates pathogens during the early stages of humoral (B-cell mediated) immunity, before sufficient levels of IgG are present. IgG is often used in immunotherapy.

[0052] The term antibody is used in its broadest sense and specifically includes, for example, single monoclonal antibodies, antibody compositions having polyepitope specificity, single-chain antibodies, and antigen-binding fragments of antibodies. Antibodies may contain immunoglobulin constant domains from any immunoglobulin, such as subtypes of IgG1, IgG2, IgG3, or IgG4, IgA (including IgA1 and IgA2), IgE, IgD, or IgM.

[0053] In some aspects, antibodies and other therapeutic molecules used herein may be isolated. To isolate, in the context of antibodies or other biological products, means that the antibody or other biological product has been removed from its natural environment or altered from its natural state. Thus, isolation does not necessarily reflect the extent to which the molecule has been removed from its natural environment or altered from its natural state. However, an antibody or other biological product that has been purified to a certain extent, or to a degree suitable for use for its intended therapeutic purpose, will be understood as “isolated.”

[0054] The antibodies used herein are humanized. Humanized forms of non-human (e.g., mouse) antibodies include chimeric immunoglobulins (including full-length immunoglobulins), immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequences of the antibody) that contain minimal sequences derived from the non-human immunoglobulin. Humanized antibodies typically contain human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are substituted with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, to possess the desired specificity, affinity, and ability. In some cases, Fv framework residues of human immunoglobulin are substituted with corresponding non-human residues. Humanized antibodies may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies will contain all or substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to the human immunoglobulin consensus sequence. Humanized antibodies optimally contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)). Modifications of the Fc region of antibodies are well-established and include modifications that cause antibodies to lose their complement-dependent cytotoxic properties and modifications that enhance the antibody's ability to cross the cell membrane.

[0055] The antibodies used herein selectively bind to their targets, and in particular to: programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), cytotoxic T lymphocyte-associated antigen (CTLA)-4, V-domain Ig suppressor of T cell activation (VISTA or PD-L3), programmed death-ligand-2 (PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase (ARG1), B7 family inhibitory ligand B7-H3, B 7 family inhibitory ligands include B7-H4, lymphocyte activator gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuators (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR), or members of the killer inhibitory receptor family, such as killer cell immunoglobulin-like receptors (KIRs) and type C lectin receptors. PD-L1 is a ligand for PD-1. CTLA-4, like PD-1, functions as an immune checkpoint molecule. VISTA is an Ig superfamily inhibitory ligand with some homology (about 25% due to sequence homology) to PD-L1 in its extracellular domain.

[0056] Aspects of this disclosure relate to the use or administration of one or more immune checkpoint inhibitors that can bind to and / or antagonize immune checkpoint molecules, such as PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, and / or KIR. Drugs that selectively bind to immune checkpoint molecules may, without limitation, be antibodies or their antigen-binding fragments, proteins or peptides, small molecules, or nucleic acids. Immune checkpoint molecules that are nucleic acids may be, for example, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNA, or siRNA. Small molecules are organic compound drugs. Drugs that selectively bind to immune checkpoint molecules may bind to nucleic acids or amino acids in the immune checkpoint molecule sequence. Drugs that selectively bind to immune checkpoint molecules can bind to any region of the immune checkpoint molecule.

[0057] Immune checkpoints. Immune checkpoints refer to immobilized inhibitory pathways in the immune system that are crucial for maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize secondary tissue damage. Immune checkpoint molecules may be stimulant or inhibitory to immune checkpoints. This disclosure and claims refer to inhibitory molecules of immune checkpoints as “immune checkpoint molecules.” Preliminary clinical findings with agents blocking immune checkpoint molecules (e.g., PD1 or CTLA-4) suggest an opportunity to enhance antitumor immunity that may produce an effective clinical response. This application discloses enhancing the efficacy of treatment in subjects with cancer or infection by combining immune checkpoint blockade with immune checkpoint inhibitors with HSCT and / or HSC mobilization treatments.

[0058] Immune checkpoint inhibitors and immune checkpoint blockades. Immune checkpoint inhibitors are a type of drug that blocks the signaling of immune checkpoint molecules produced by certain types of immune system cells, such as T cells and some cancer cells. Therefore, immune checkpoint inhibitors can cause immune checkpoint blockade. Immune checkpoint molecules (e.g., PD1) help maintain the checks on the immune response, allowing T cells to prevent them from killing cancer cells. When these molecules are blocked, the "brakes" on the immune system are released (immune system inhibition is reduced or blocked), allowing T cells to kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4. In some aspects, immune checkpoint molecules are proteins. In some aspects, immune checkpoint molecules are nucleic acids that encode proteins. In some aspects, immune checkpoint inhibitors bind to and / or antagonize immune checkpoint molecules. In some aspects, immune checkpoint inhibitors are used in combination with hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilization treatments to treat subjects with cancer. In some embodiments, immune checkpoint inhibitors are used in combination with hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilization treatment to treat subjects with infections.

[0059] As described above, according to the present invention, immune checkpoint blockade is used in combination therapy with hematopoietic stem cell (HSC) transplantation / implantation and / or HSC mobilization. In some embodiments, methods for treating a disease or a subject having the disease include administering HSCs and / or HSC mobilizers, and administering agents that bind to and / or antagonize programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and / or V-domain Ig suppressor of T cell activation (VISTA). The present disclosure is not limited to targeting PD-1, PD-L1, CTLA-4, and / or VISTA for immune checkpoint blockade. Other inhibitory checkpoint molecules may also be targeted by immune checkpoint inhibitors in combination therapy with HSC transplantation and / or HSC recruitment, including, but are not limited to, agents that bind to and / or antagonize the following: programmed death ligand 2 (PD-L2), indoleamine 2,3-deoxygenase (IDO), arginase (ARG1), B7 family inhibitory ligand B7-H3, B7 family inhibitory ligand B7-H4, lymphocyte activator gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR), members of the killer inhibitory receptor family (KIR), such as killer cell immunoglobulin-like receptors (KIR) and type C lectin receptors, as well as signaling and transcriptional activators (STAT3). In some embodiments, immune checkpoint molecules are, for example, PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, STAT3, or KIR. Drugs that bind to and / or antagonize immune checkpoint molecules are immune checkpoint inhibitors.

[0060] One or more immune checkpoint inhibitors. One or more immune checkpoint inhibitors refer to one or more different inhibitors. Each different inhibitor has a different molecular structure. Two different inhibitors may bind to the same immune checkpoint molecule, or each may bind to a different immune checkpoint molecule. The inhibitors or antagonists used herein are molecules that inhibit, reduce, or block the activity of immune checkpoint molecules, thereby inhibiting the suppressive effect that immune checkpoint molecules have on the immune system. Inhibitors or antagonists can directly bind to immune checkpoint molecules, molecules that regulate the expression of immune checkpoint molecules, or ligands of immune checkpoint molecules that mediate the activity of immune checkpoint molecules. Inhibitors or antagonists may be antibodies (including humanized antibodies), small molecules, peptides, or nucleic acids (e.g., antisense molecules, or single-stranded or double-stranded RNAi molecules). The activity of an immune checkpoint molecule is defined as its suppressive effect on immune checkpoints. Immune checkpoint inhibitors can reduce or block the activity of immune checkpoint molecules.

[0061] Exemplary immune checkpoint molecules and antagonists. Programmed Death 1 (PD-1). In humans, programmed cell death protein 1 (PD-1) is encoded by the PDCD1 gene. PDCD1 is also designated as CD279 (differentiation cluster 279). This gene encodes a cell surface membrane protein of the immunoglobulin superfamily. PD-1 is a 288-amino acid cell surface protein molecule. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages. PD-1 is expressed in pro-B cells and is thought to play a role in their differentiation. See T. Shinohara et al., Genomics 23 (3): 704-6 (1995). PD-1 is a member of the expanded CD28 / CTLA-4 family of T cell regulators. (Y. Ishida et al., "EMBO J. 11 (11): 3887-95, (1992)). PD-1 can negatively modulate the immune response. PD-1 restricts T cell activity in autoimmune and peripheral tissues during inflammatory responses to infection.

[0062] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, as well as on T cells and B cells during TCR and B cell receptor signaling. In resting mice, however, PD-L1 mRNA can be detected in the heart, lungs, thymus, spleen, and kidneys. PD-L1 is expressed in almost all mouse tumor cell lines, including PA1 myeloma, P815 mast cell tumor, and B16 melanoma, upon treatment with IFN-γ. PD-L1 has been found to be highly expressed by several cancers, and several PD-1 antagonists are being developed or approved for cancer treatment. PD-L2 expression is more restricted and is expressed primarily by DCs and some tumor lines.

[0063] Programme Death 1 (PD-1) Antagonists. As used herein, PD-1 antagonists are molecules that bind to the PD-1 protein or the gene or nucleic acid encoding the PD-1 protein, thereby inhibiting or preventing PD-1 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between PD-1 and its ligands PD-L1 and / or PD-L2. In some embodiments, PD-1 antagonists, when administered in combination with HSCT and / or HSC mobilization agent treatment, may reduce PD-1 activity in cells or organisms compared to cells or organisms not exposed to the PD-1 antagonist, more so than when administered alone.

[0064] PD-1 activity can be inhibited by antibodies that selectively bind to PD-1 and block its activity. PD-1 activity can also be inhibited or blocked by molecules other than antibodies that bind to PD-1. Such molecules may be small molecules or peptide mimes of PD-L1 and PD-L2 that bind to PD-1 but do not activate it. Examples of molecules that antagonize PD-1 activity are described in U.S. Patent Publications 20130280265, 20130237580, 20130230514, 20130109843, 20130108651, 20130017199, and 20120251537, 2011 / 0271358, EP 2170959B1, the entirety of which is incorporated herein by reference. See also MA Curran, et al., Proc. Natl. Acad. Sci. USA 107, 4275 (2010); SL ​​Topalian, et al., New Engl. J. Med. 366, 2443 (2012); JR Brahmer, et al., New Engl. J. Med. 366, 2455 (2012); and DE Dolan et al., Cancer Control 21, 3 (2014), all of which are incorporated herein by reference in their entirety.In this specification, exemplary PD-1 antagonists include: nivolumab, also known as BMS-936558, OPDIVO® (also known as Bristol-Meyers Squibb, and MDX-1106 or ONO-4538), which is a fully human IgG4 monoclonal antibody against PD-1; pizilizumab, also known as CT-011 (CureTech), which is a humanized IgG1 monoclonal antibody that binds to PD-1; and MK-3475 (Merck, and SCH PD-1-binding IgG4 antibody (also known as 900475); and pembrolizumab (Merck, and also known as MK-3475, lambrolizumab, or KEYTRUDA®), a humanized IgG4κ monoclonal antibody that binds to PD-1; MEDI-0680 (AstraZeneca / MedImmune), a monoclonal antibody that binds to PD-1; REGN2810 (Regeneron / Sanofi), a monoclonal antibody that binds to PD-1. Another exemplary PD-1 antagonist is AMP-224 (Glaxo Smith Kline and Amplimmune), a recombinant fusion protein composed of the extracellular domain of programmed cell death ligand 2 (PD-L2), a PD-1 ligand, and the Fc region of human IgG1, which binds to PD-1. Drugs that prevent binding to the DNA or mRNA encoding PD-1 can also act as PD-1 inhibitors. Examples include small inhibitory anti-PD-1 RNAi, anti-PD-1 antisense RNA, or dominant-negative proteins. The PDL-2 fusion protein AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune) is thought to bind to and block PD-1. In some embodiments, anti-PD-1 antibodies may be used in combination with hematopoietic stem cell (HSC) transplantation and / or HSC recruitment, or in further combination with additional immune checkpoint blockade such as anti-PD-L1, anti-CTLA-4, and / or anti-VISTA treatment.

[0065] Programmed death-ligand 1 (PD-L1), also known in humans as B7 homolog 1 (B7-H1) or differentiation cluster 274 (CD274), is a 40 kDa type 1 transmembrane protein encoded by the CD274 gene. Foreign antigens typically induce an immune response that causes the proliferation of antigen-specific T cells, such as antigen-specific CD8+ T cells. PD-L1 is an immune checkpoint inhibitor that blocks or reduces such immune responses. PD-L1 can play a major role in suppressing the immune system during events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other disease conditions such as hepatitis and cancer. The PD-L1 ligand binds to its receptor PD-1, found on activated T cells, B cells, and myeloid cells, thereby regulating activation or inhibition. In addition to PD-1, PD-L1 also has affinity for the costimulatory molecule CD80 (B7-1). Upon IFN-γ stimulation, PD-L1 is expressed on T cells, natural killer (NK) cells, macrophages, myeloid dendritic cells (DCs), B cells, epithelial cells, and vascular endothelial cells.

[0066] PD-L1 Antagonists. As used herein, PD-L1 antagonists are molecules that bind to the PD-L1 protein or the gene or nucleic acid encoding the PD-L1 protein, thereby inhibiting or preventing PD-1 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between PD-L1 and PD-1. PD-L1 activity can be blocked by molecules that selectively bind to PD-L1 and block its activity. In some embodiments, PD-L1 antagonists, when administered in combination with HSCT and / or HSC mobilization agent treatment, may reduce PD-L1 activity in cells or organisms compared to cells or organisms not exposed to the PD-1 antagonist, compared to when administered alone. Anti-PD-L1 antibodies block the interaction between PD-L1 and PD-1 and B7-1 (also known as CD80). Blockade means inhibiting or preventing the transmission of inhibitory signals mediated through such PD-L1 binding. Examples of PD-L1 antagonists include: BMS-936559, also known as MDX-1105 (Bristol-Meyers Squibb), a fully human high-affinity immunoglobulin (Ig) G4 monoclonal antibody against PD-L1; MPDL3280A, also known as RG7446 or atezolizumab (Genentech / Roche), a modified human monoclonal antibody that targets PD-L1; MSB0010718C, also known as avelumab (Merck), a fully human IgG1 monoclonal antibody that binds to PD-L1; and MEDI473 (AstraZeneca / MedImmune), a human immunoglobulin (Ig) G1κ monoclonal antibody that blocks PD-L1 binding to its receptor. Drugs that bind to the DNA or mRNA encoding PD-L1 can also act as PD-L1 inhibitors, such as small inhibitory anti-PD-L1 RNAi, small inhibitory anti-PD-L1 RNA, anti-PD-L1 antisense RNA, or dominant-negative PD-L1 protein.Antagonists or agents that antagonize PD-L1, such as PD-L1 antibodies or PD-L1 antagonists, may include, but are not limited to, those described above and any of those disclosed in Stewart et al., 2015, 3(9):1052-62; Herbst et al., 2014, Nature Volume: 515:Pages: 563-567; Brahmer et al., N Engl J Med 2012; 366:2455-2465; US8168179; US20150320859; and / or US20130309250; all of which are incorporated herein by reference. In clinical trials, treatment with anti-PD-L1 antibodies resulted in fewer adverse events than treatment with anti-PD-1 antibodies (Shih et al., 2014). In some embodiments, anti-PD-L1 antibodies may be used for treatment in combination with hematopoietic stem cell (HSC) transfer and / or HSC mobilization, in further combination with additional immune checkpoint blockade such as anti-PD-1, anti-CTLA-4, and / or anti-VISTA treatment.

[0067] Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). CTLA-4 (also known as CTLA-4 or differentiation cluster 152 (CD152)) is a transmembrane glycoprotein encoded by the CTLA-4 gene in humans. CTLA-4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells and present on regulatory T cells, and can be important for immune function. CTLA-4, like its homologous CD28, binds to B7 molecules, particularly CD80 / B7-1 and CD86 / B7-2 on antigen-presenting cells (APCs), thereby sending inhibitory signals to T cells. CTLA-4 functions as an immune checkpoint that inhibits the immune system and is important for maintaining immune tolerance.

[0068] CTLA-4 antagonists. As used herein, CTLA-4 antagonists are molecules that bind to the CTLA-4 protein or the gene or nucleic acid encoding the CTLA-4 protein to inhibit or prevent CTLA-4 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between CTLA-4 and its ligands, such as the B7 molecules CD80 / B7-1 and CD86 / B7-2. CTLA-4 activity can be blocked by molecules that selectively bind to CTLA-4 and block its activity, or by molecules that selectively bind to its counterreceptors, such as CD80, CD86, etc., and block CTLA-4 activity. Blocking means inhibiting or preventing the transmission of inhibitory signals mediated by CTLA-4. In some embodiments, anti-CTLA-4 antibodies may be used for treatment in combination with hematopoietic stem cell (HSC) transfer and / or HSC mobilization, in further combination with additional immune checkpoint blockade, such as anti-PD-1, anti-CTLA-4, and / or anti-VISTA treatment. Examples of CTLA-4 antagonists include inhibitory antibodies against CD80, CD86, and / or CTLA-4; small molecule inhibitors of CD80, CD86, and CTLA-4; antisense molecules against CD80, CD86, and / or CTLA-4; adnectin against CD80, CD86, and / or CTLA-4; and RNAi inhibitors (both single-stranded and double-stranded) of CD80, CD86, and / or CTLA-4.

[0069] Suitable CTLA-4 antagonists and / or anti-CTLA-4 antibodies include humanized anti-CTLA antibodies, e.g., MDX-010 / ipilimumab (Bristol-Meyers Squibb), tremelimumab / CP-675,206 (Pfizer; AstraZeneca), and the antibodies described below: PCT Publication WO 2001 / 014424, PCT Publication WO 2004 / 035607, US Publication 2005 / 0201994, European Patent 1212422. U.S. Patent Nos. B1, 5,811,097, 5,855,887, 6,051,227, 6,984,720, 7,034,121, 8,475,790, U.S. Publication No. 2002 / 0039581 and / or 2002 / 086014; all of these disclosures are incorporated herein by reference. Other anti-CTLA-4 antibodies and / or CTLA-4 antagonists that can be used in the methods of this disclosure include, for example, those listed below: Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and Lipson and Drake, Clin Cancer Res; 17(22) November 15, 2011; US8318916; and / or EP1212422B1; all of which are incorporated herein by reference in their entirety.

[0070] The V-domain Ig suppressor of T cell activation (VISTA). The V-domain Ig suppressor of T cell activation (VISTA) (also known as PD-H1, PD-1 homolog, or Dies1) is a negative regulator of T cell function. VISTA is a 309aa type I transmembrane protein consisting of seven exons, possessing one Ig-V-like domain whose sequence is similar to that of the Ig-V domains of members of the CD28 and B7 families. VISTA is highly expressed in the tumor microenvironment (TME) and hematopoietic cells. It is also expressed in macrophages, dendritic cells, neutrophils, natural killer cells, and naive and activated T cells. Its expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells, while low levels are associated with CD4 + T cells, CD8 + T cells and T reg It is found on cells. VISTA shows some sequence homology to PD-L1, a PD-1 ligand, but these two immune checkpoint inhibitors are structurally different and have different signaling pathways. Blocking VISTA has been shown to enhance the antitumor immune response in mice, while in humans, blocking the associated PD-1 pathway shows great potential in clinical immunotherapy trials. VISTA is a negative checkpoint modulator that suppresses T cell activation, and its blockade may be an effective immunotherapy strategy against human cancer. (Wang et al., 2011. JEM. 208(3):577-92.; Lines et al., 2014. Cancer Res. 74(7):1924-32.; Kondo et al. 2015. J. of Immuno.V194.; WO2011120013; US20140105912; US20140220012; US20130177557, US20130177557; these in their entirety are incorporated herein by reference).

[0071] VISTA antagonists. As used herein, VISTA antagonists are molecules that bind to the VISTA protein or the gene or nucleic acid encoding the VISTA protein, thereby inhibiting or preventing VISTA activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between VISTA and its ligand(s). VISTA activity can be blocked by molecules that selectively bind to VISTA and block its activity. Molecules or drugs that are VISTA antagonists include peptides that bind to VISTA, antisense molecules against VISTA, single-stranded or double-stranded RNAi molecules targeted to degrade or inhibit VISTA, small molecule inhibitors of VISTA, anti-VISTA antibodies, inhibitory antibodies against VISTA, and humanized antibodies that selectively bind to and inhibit VISTA. VISTA antagonists or drugs that antagonize VISTA, such as anti-VISTA antibodies and VISTA antagonists, may include, but are not limited to, any of the following: Liu et al. 2015. PNAS. 112(21):6682-6687; Wang et al., 2011. JEM. 208(3):577-92; Lines et al., 2014. Cancer Res. 74(7):1924-32; Kondo et al. 2015. J. of Immuno.V194; WO2015097536, EP2552947, WO2011120013, US20140056892, US8236304. WO2014039983, US20140105912, US20140220012, US20130177557; WO2015191881; US20140341920; CN105246507; and / or US20130177557, all of which are incorporated herein by reference in their entirety. In some embodiments, anti-VISTA antibodies may be used for treatment in combination with hematopoietic stem cell (HSC) transfer and / or HSC mobilization, in further combination with additional immune checkpoint blockade such as anti-PD-1, anti-CTLA-4, and / or anti-VISTA treatment.

[0072] Other immunosuppressive molecules and immune checkpoint inhibitors. Molecules other than PD-1, PD-L1, CTLA-4, and VISTA may be targeted by one or more immune checkpoint inhibitors / agents that bind to and / or antagonize immune checkpoint molecules, in combination with treatment of hematopoietic stem cells and / or hematopoietic stem cell mobilizers. In some embodiments, one or more immune checkpoint inhibitors are antagonists of PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, KIR, and / or STAT3.

[0073] Programmed death ligand 2 (PD-L2). Human PD-L2, also known as the B7 dendritic cell molecule B7-DC, Btdc, PD-1 ligand 2, PDCD1 ligand 2, butyrophyllin B7-DC, and bA574F11.2, is a protein encoded by the PDCD1LG2 gene (also known as differentiation cluster 273 / CD273). PD-L2 is an inhibitory molecule primarily expressed via Th2-related cytokines in a manner that can be induceable by antigen-presenting cells, T cells and other immune cells, and some non-immune cells. For further information, see Rozali et al., Clinical and Developmental Immunology, 2012 (2012). PD-L2 involvement of PD-1 is linked to T cell receptor (TCR)-mediated proliferation and CD4 + It dramatically inhibits cytokine production by T cells. At low antigen concentrations, the PD-L2-PD-1 interaction inhibits potent B7-CD28 signaling. In contrast, at high antigen concentrations, the PD-L2-PD-1 interaction reduces cytokine production but does not inhibit T cell proliferation (Latchman et al., Nature Immunology, 2(3):261-268 (2001)). As used herein, PD-L2 antagonists are molecules that bind to the PD-L2 protein or the gene or nucleic acid encoding the PD-L2 protein, thereby inhibiting or preventing PD-L2 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between PD-L2 and its ligand(s) (e.g., PD-1).

[0074] Indoleamine 2,3-dioxygenase (IDO). Also known as INDO and IDO-1, this is the gene that encodes indoleamine 2,3-dioxygenase (IDO) (a heme enzyme that catalyzes the first and rate-limiting steps of the catabolism of tryptophan to N-formyl-kynurenine). IDO is a cytosolic metabolic enzyme, an immunosuppressive molecule expressed by tumor cells and infiltrating myeloid cells. The IDO enzyme is overexpressed by various tumor cell types and antigen-presenting cells (APCs) and is involved in tryptophan catabolism and the conversion of tryptophan to kynurenine. This enzyme acts on multiple tryptophan substrates, including D-tryptophan, L-tryptophan, 5-hydroxytryptophan, tryptamine, and serotonin. This enzyme is thought to play a role in various pathophysiological processes, such as antimicrobial and antitumor defense, neuropathology, immunomodulation, and antioxidant activity. Through its expression in dendritic cells, monocytes, and macrophages, this enzyme regulates T cell behavior by pericellular catabolism of the essential amino acid tryptophan. The IDO enzyme can be inhibited to enhance intratumoral inflammation by molecular analogs of its substrate that act as competitive inhibitors or suicide substrates. The suppressed activation of the immune system in many cancers can induce a cytotoxic T lymphocyte (CTL) response to IDO1-expressing tumor cells. Tryptophan depletion inhibits T lymphocyte proliferation and activation and is associated with immunosuppression caused by T cell suppression.

[0075] As used herein, IDO antagonists are molecules that bind to the IDO protein or the gene or nucleic acid encoding the IDO protein, thereby inhibiting or preventing IDO activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block IDO activity. Exemplary IDO antagonists include, for example, the IDO inhibitors epacadostat, NLG919, and indoleamine 2,3-dioxygenase peptide vaccine.

[0076] Epacadostat is an orally available hydroxyamidine and indoleamine 2,3-dioxygenase (IDO1) inhibitor with potential immunomodulatory and antitumor activity. Epacadostat targets and binds to the enzyme IDO1, which is responsible for the oxidation of tryptophan to kynurenine. By inhibiting IDO1 and reducing kynurenine in tumor cells, epacadostat increases and restores the proliferation and activation of various immune cells, including dendritic cells (DCs), NK cells, and T lymphocytes, as well as interferon (IFN) production and tumor-associated regulatory T cells (Tregs).

[0077] NLG919 is an orally available inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) with potential immunomodulatory and antitumor activity. Upon administration, NLG919 targets and binds to IDO1, a cytosolic enzyme responsible for the oxidation of the essential amino acid tryptophan to kynurenine. By inhibiting IDO1 and reducing kynurenine in tumor cells, the drug increases tryptophan levels, restoring the proliferation and activation of various immune cells, including dendritic cells (DCs), natural killer (NK) cells, and T lymphocytes, and causing a reduction in tumor-associated regulatory T cells (Tregs).

[0078] Indoleamine 2,3-dioxygenase peptide vaccine is a peptide vaccine targeting the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) and possesses potential immunomodulatory and antitumor activity. Vaccination with indoleamine 2,3-dioxygenase peptide vaccine can activate the immune system and induce an immune response against IDO-expressing cells. This can increase and restore the proliferation and activation of various immune cells, including dendritic cells (DCs), natural killer (NK) cells, and T lymphocytes, potentially eradicating IDO-expressing tumor cells.

[0079] Arginase (ARG1). ARG1 is another metabolic enzyme that is an immunosuppressive molecule. It is produced by bone marrow-derived suppressor cells. Arginase catalyzes the hydrolysis of arginine to ornithine and urea. There are at least two isoforms of mammalian arginase (type I and type II), which differ in tissue distribution, subcellular localization, immunological cross-reactivity, and physiological function. The type I isoform encoded by this gene is a cytosolic enzyme and is mainly expressed in the liver as a component of the urea cycle. Genetic deficiency of this enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. Two transcriptional variants encoding different isoforms have been found for this gene.

[0080] As used herein, ARG1 antagonists are molecules that bind to the ARG1 protein or the gene or nucleic acid encoding the ARG1 protein, thereby inhibiting or preventing ARG1 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block ARG1 activity. Arginase enzymes can be inhibited to enhance intratumoral inflammation by molecular analogs of their substrates that act as competitive inhibitors or suicide substrates. B7 family inhibitory ligands B7-H3 and B7-H4. B7 family members and their known ligands belong to the immunoglobulin superfamily. The B7 family has co-stimulatory and inhibitory receptors. Numerous B7 family inhibitory ligands, such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1), do not yet have defined receptors, but mouse knockout experiments support the immunoinhibitory role of these ligands. B7-H3 and B7-H4 are upregulated on tumor cells or tumor-infiltrating cells. B7-H3 may be upregulated on endothelial cells of tumor vascular structures, and B7-H4 has been reported to be expressed on tumor-associated macrophages. B7-H4 is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape. Preclinical mouse models of cancer have shown that blocking ligands or receptors of many individual immune checkpoint B7 family members can enhance antitumor immunity, and that double blocking of synergistically expressed receptors results in additive or synergistic antitumor activity. Numerous inhibitors / antagonists for these immune checkpoint targets are either in clinical practice or actively under development.

[0081] As used herein, B7 family member antagonists are molecules that bind to B7 family member proteins or genes or nucleic acids encoding B7 family member proteins, thereby inhibiting or preventing the activation of B7 family members. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between B7 family members, such as B7-H3 or B7-H4, and their ligand(s). Exemplary B7-H3 antagonists include, for example, enobrituzumab (MacroGenics, also known as MGA271), an Fc-optimized monoclonal antibody targeting B7-H3, and MGD009 (MacroGenics), a biaffinity retargeting (DART®) molecule targeting B7-H3 and CD3. Blocking antibodies or small molecule inhibitors are currently available for molecules that can be targeted by agents that bind to and / or antagonize, for example, LAG3, 2B4, BTLA, TIM3, A2aR, and the killer inhibitory receptor family, in the context of this disclosure.

[0082] Lymphocyte-activating gene 3 (LAG3). LAG3 (also known as CD233) belongs to the immunoglobulin superfamily (IgSF) and contains four extracellular Ig-like domains. It binds to major histocompatibility complex (MHC) class II. LAG-3 expression on tumor-infiltrating lymphocytes (TILs) is associated with tumor-mediated immunosuppression. LAG3 is associated with T Reg It has been shown to play a role in enhancing cell function. LAG3 is T RegIndependent of its role for cells, it inhibits CD8+ effector T cell function. A known ligand of LAG3 is major histocompatibility complex (MHC) class II. MHC class II molecules are upregulated generally in response to IFNγ in some epithelial cancers and are expressed on tumor-infiltrating macrophages and dendritic cells. The role of the LAG3-MHC class II interaction in the LAG3-mediated inhibition of T cell responses is unclear because LAG3 antibodies that do not block the LAG3-MHC class II interaction still enhance T cell proliferation and effector cell function in vitro and in vivo. This interaction may be most important for the role of LAG3 in enhancing T Reg cell function. LAG3 is one of various immune checkpoint receptors that are coordinately upregulated in both T Reg cells and anergic T cells, and the simultaneous blockade of these receptors can enhance the reversal of this anergic state compared to the blockade of only one receptor. In particular, PD1 and LAG3 are generally co-expressed on anergic or exhausted T cells. Dual blockade of LAG3 and PD1 synergistically reversed anergy between tumor-specific CD8+ T cells and virus-specific CD8+ T cells in the setting of chronic infection.

[0083] As used herein, LAG3 antagonists are molecules that bind to the LAG3 protein or the gene or nucleic acid encoding the LAG3 protein, thereby inhibiting or preventing LAG3 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between LAG3 and its ligand(s). An exemplary LAG3 antagonist is BMS-986016 (Bristol-Myers Squibb), a monoclonal antibody that binds to LAG-3 and possesses potential immune checkpoint inhibitory and antitumor activity. Upon administration, BMS-986016 binds to LAG-3 on tumor-infiltrating lymphocytes (TILs). This activates antigen-specific T lymphocytes, enhancing cytotoxic T cell-mediated tumor cytolysis and resulting in reduced tumor growth. 2B4 (also known as CD244 and SLAMf4) is a 38kD type I transmembrane protein and a member of the CD2 subset of the immunoglobulin superfamily (Lee et al., 2004; Vaidya et al., 2005). It is encoded by the gene (2B4) which encodes a cell surface receptor expressed on natural killer (NK) cells and some T cells that mediate non-major histocompatibility complex (MHC) restricted death. Interactions between NK cells and target cells via the 2B4 receptor are thought to regulate the cytolytic activity of NK cells. 2B4 is a co-inhibitory molecule that has been identified as being expressed in cells removed after chronic viral infection. It is expressed on NK cells, monocytes, basophils, and eosinophils and is inductively expressed on a subset of CD8+ T cells in both mouse and human (see Liu et al., JEM, 211(2):297-311 (2014) and its references). Alternatively, transcriptional splice variants encoding different isoforms have been found for the human 2B4 gene.

[0084] B and T lymphocyte attenuator (BTLA). BTLA (also known as B and T lymphocyte-associated, BTLA-1, and CD272) is a gene encoding a member of the immunoglobulin superfamily. The encoded protein contains a single immunoglobulin (Ig) domain and is a receptor that transmits inhibitory signals and suppresses the immune response. Alternative splicing results in multiple transcriptional variants. Polymorphisms in this gene are associated with an increased risk of rheumatoid arthritis. BTLA was initially identified as an inhibitory receptor on T cells based on the enhanced T cell response observed in Btla knockout mice. Subsequently, herpesvirus entry mediator (HVEM; also known as TNFRSF14), expressed on certain tumor cell types (e.g., melanoma) and tumor-associated endothelial cells, was shown to be a BTLA ligand. This is a rare case of a TNF family member interacting with an immunoglobulin supergene family member. BTLA expression levels are quite low in activated virus-specific CD8+ T cells, but can be very high in tumor-infiltrating lymphocytes (TILs) from melanoma patients. hi T cells are inhibited in the presence of their ligand, HVEM. Therefore, BTLA may be an associated inhibitory receptor for T cells in the tumor microenvironment. As used herein, BTLA antagonists are molecules that bind to the BTLA protein or the gene or nucleic acid encoding the BTLA protein, thereby inhibiting or preventing BTLA activation. While we do not wish to be constrained by theory, such molecules are thought to reduce or block the interaction between BTLA and its ligand(s).

[0085] T cell membrane protein 3 (TIM3). The TIM3 gene (also known as hepatitis A virus cell receptor 2 / HAVcr2, CD366, KIM-3, TIMD3, Tim-3, and TIMD-3) encodes proteins belonging to the immunoglobulin superfamily and the TIM family. CD4-positive T helper lymphocytes can be divided into type 1 (Th1) and type 2 (Th2) based on their cytokine secretion patterns. Th1 cells are involved in cell-mediated immunity against intracellular pathogens and delayed-type hypersensitivity reactions, while Th2 cells are involved in the control of extracellular parasitic infections and the promotion of atopic and allergic diseases. The TIM3 protein is a Th1-specific cell surface protein that regulates macrophage activation, inhibits Th1-mediated autoimmune responses, and promotes immunological tolerance. TIM3, whose ligand is galectin 9 (a galectin upregulated in various types of cancer, including breast cancer), inhibits the T helper 1 (TH1) cell response, and TIM3 antibodies enhance antitumor immunity. TIM3 has been reported to be co-expressed with PD1 on tumor-specific CD8+ T cells, and double blockade of both molecules significantly enhances in vitro proliferation and cytokine production of human T cells when stimulated by cancer-testis antigen, NY-ESO-1. In animal models, coordinated blockade of PD1 and TIM3 has been reported to enhance antitumor immune responses and tumor rejection in situations where blockade of each individual molecule has only a slight effect. As used herein, TIM3 antagonists are molecules that bind to the TIM3 protein or the gene or nucleic acid encoding the TIM3 protein, thereby inhibiting or preventing TIM3 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between TIM3 and its ligand(s).

[0086] The adenosine A2a receptor (A2aR). The A2aR gene (also known as RDC8 and ADORA2) encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily, which is subdivided into classes and subtypes. Receptors are 7-path transmembrane proteins that activate intracellular signaling pathways in response to extracellular cues. A2aR is a G protein-coupled receptor that is highly expressed on the cell surface of T cells and inhibits their proliferation and activation upon adenosine activation. Adenosine is often overproduced by cancer cells. The A2aR protein, an adenosine (ligand) receptor of the A2A subtype, uses adenosine as a preferred endogenous agonist and preferentially interacts with the G(s) and G(olf) families of G proteins to increase intracellular cAMP levels. This plays a crucial role in many biological functions, such as cardiac rhythm and circulation, cerebral and renal blood flow, immune function, pain regulation, and sleep. This is involved in pathophysiological conditions such as inflammatory and neurodegenerative diseases. Alternative splicing results in multiple transcriptional variants. Readthrough transcripts consisting of upstream SPECC1L (sperm antigen with calponin homology and coiled-coil domain 1-like) and ADORA2A (adenosine A2a receptor) gene sequences have been identified but are thought to be non-coding. A2aR inhibits the T cell response, partly by driving CD4+ T cells to express FOXP3 and thereby develop into T cells. Deficiency of this receptor results in an enhanced, sometimes pathological, inflammatory response to infection. The A2aR receptor is particularly relevant to tumor immunity because tumors have a high rate of cell death from cell turnover, and dying cells release adenosine. Furthermore, T Reg The cells express high levels of the extracellular enzyme CD39 (also known as NTPDase1), which converts extracellular ATP to AMP, and CD73 (also known as 5'-NT), which converts AMP to adenosine. Adenosine-mediated A2aR involvement drives T cells. RegConsidering its cellular nature, this can generate self-amplifying loops within tumors. Tumors grow more slowly in A2aR (also known as Adora2a) knockout mice, and tumor vaccines are far more effective against established tumors in these mice.

[0087] As used herein, A2aR antagonists are molecules that bind to the A2aR protein or the gene or nucleic acid encoding the A2aR protein, thereby inhibiting or preventing A2aR activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between A2aR and its ligand(s). A2aR can be inhibited by either antibodies that block adenosine binding or adenosine analogs (some of which are quite specific to A2aR). These drugs have been used in clinical trials for Parkinson's disease but have not been clinically tested in cancer patients. An exemplary A2aR antagonist, to some extent, is the adenosine A2A receptor antagonist PBF-509. PBF-509 is an orally bioavailable adenosine A2A receptor (A2AR) antagonist with potential antitumor activity. Upon administration, the A2AR antagonist PBF-509 selectively binds to and inhibits A2AR expressed on T lymphocytes. This neutralizes the adenosine / A2AR-mediated inhibition of T lymphocytes, activating the T cell-mediated immune response against tumor cells and thereby reducing the proliferation of susceptible tumor cells.

[0088] Killer inhibitory receptors (KIRs). Members of the immunoglobulin superfamily, KIRs are expressed on the surface of NK cells. Killer cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by subsets of natural killer cells and T cells. KIR genes are polymorphic, highly homologous, and found in clusters on chromosome 19q13.4 within the 1Mb leukocyte receptor complex (LRC). Gene content of KIR gene clusters differs among haplotypes, however, some "framework" genes are found in all haplotypes (KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2). KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and whether they have long (L) or short (S) cytoplasmic domains. KIR proteins with long cytoplasmic domains transmit inhibitory signals upon ligand binding via an immunotyrosine-based inhibitory motif (ITIM), while KIR proteins with short cytoplasmic domains lack the ITIM motif and instead associate with TYRO protein tyrosine kinase-binding proteins to transmit activation signals. KIR is a broad category of inhibitory receptors that can be classified into two classes based on structure: killer cell immunoglobulin-like receptors (KIRs) and type C lectin receptors, both of which are type II transmembrane receptors. These receptors were initially described as important regulators of NK cell death activity, but many are expressed on T cells and APCs. The importance of their inhibitory role on T cells and APCs (e.g., dendritic cells) has not been studied much, but the resulting NK cell activation can provide potent antitumor activity. Many killer inhibitory receptors are specific to a subset of human leukocyte antigens (HLAs; human MHC molecules) and exhibit allele specificity. However, other receptors recognize widely expressed molecules; for example, the C-type lectin receptor KLRG1 recognizes E-cadherin.The potential value of NK cells in antitumor immune responses when these inhibitory receptors are not properly involved is best exemplified by the significant enhancement of the graft-versus-tumor effect in allogeneic bone marrow transplantation, induced by a mismatch between donor NK inhibitory receptors and recipient HLA alleles. Ligands for several KIR proteins are subsets of HLA class I molecules; therefore, KIR proteins are thought to play a crucial role in regulating immune responses. Antagonists of any killer inhibitory receptor may be used in the context of this disclosure.

[0089] As used herein, KIR antagonists are molecules that bind to the KIR protein or the gene or nucleic acid encoding the KIR protein, thereby inhibiting or preventing KIR activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between KIR and its ligand(s). An exemplary KIR antagonist is lirirumab (Bristol-Myers Squibb), a fully humanized monoclonal antibody against killer cell immunoglobulin-like receptors (KIRs) with potential immune checkpoint inhibitory and antitumor activity. Upon administration, lirirumab binds to KIR, thereby preventing the binding of KIR ligands to KIR on natural killer (NK) cells. By blocking these inhibitory receptors, NK cells are activated to attack cancer cells, leading to tumor cell death.

[0090] Signaling and transcriptional activator (STAT3). Proteins encoded by the STAT3 gene are members of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases, then translocate to the cell nucleus to form homodimers or heterodimers, where they act as transcriptional activators. Seven members of the STAT protein family have been identified in mammals: STAT1, 2, 3, 4, 5a, 5b, and 6. All family members share six distinct structural domains: N-terminus, coiled-coil, DNA-binding, Src homology 2 (SH2), and transactivation domains, and contain a critical tyrosine (Tyr) residue at the C-terminus (Tyr705 in the case of STAT3), which is phosphorylated during activation. STAT3 proteins are activated by phosphorylation in response to various cytokines and growth factors, including IFN, EGF, IL5, IL6, HGF, LIF, and BMP2. This protein mediates the expression of various genes in response to cellular stimuli and therefore plays a crucial role in many cellular processes, including cell proliferation and apoptosis. The small GTPase Rac1 has been shown to bind to this protein and regulate its activity. The PIAS3 protein is a specific inhibitor of this protein. Mutations in this gene are associated with infant-onset multisystem autoimmune disease and hyperimmune globulin E syndrome. Alternative splicing results in multiple transcriptional variants encoding different isoforms.

[0091] As used herein, STAT3 antagonists are molecules that bind to the STAT3 protein or the gene or nucleic acid encoding the STAT3 protein, thereby inhibiting or preventing STAT3 activation. While we do not wish to be bound by theory, such molecules are thought to reduce or block the interaction between STAT3 and its ligand(s). Examples of small molecule compounds that are STAT3 antagonists include NSC74859(S3I-201), NSC42067, NSC59263, NSC75912, NSC11421, NSC91529, and NSC263435 (see U.S. Patent No. 7960434 B2). Many other examples of STAT3 inhibitors / antagonists can be found in Yue and Turkson, Expert Opin Investig Drugs, 2009, 18(1):45-56, which is incorporated herein by reference.

[0092] Antagonists of the inhibitory immune checkpoint molecules described may be used in the context of this disclosure. Antagonists of other molecules that are inhibitory immune molecules may be used in the context of this disclosure. In some embodiments, an antagonist of any immunosuppressive molecule may be used in combination with hematopoietic stem cells and / or hematopoietic stem cell mobilizers for the treatment of subjects having a disease, where the disease is cancer or an infection. In some embodiments, antagonists of PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7-H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, KIR, and / or STAT3 may be used in combination with hematopoietic stem cell (HSC) transfer and / or HSC mobilization treatments, in further combination with additional immune checkpoint blockade, such as anti-PD-1, anti-CTLA-4, and / or anti-VISTA treatments.

[0093] Hematopoietic stem cells (HSCs), also known as blood stem cells, are immature cells found in the blood and bone marrow that can regenerate themselves and differentiate into various specialized cells, including blood and immune cells such as white blood cells, red blood cells, and platelets. HSCs can be recruited from the bone marrow into the circulating blood. HSCs promote the continuous regeneration of blood cells, producing billions of new blood cells every day.

[0094] Hematopoietic stem cell transplantation (HSCT). Hematopoietic stem cell (HSC) transplantation (HSCT or HSC transfer) is the transplantation of HSCs, usually derived from peripheral blood, bone marrow, or umbilical cord blood. Two types of HSCT can be used for the subject: autologous stem cell transplantation, which uses the subject's own stem cells; or allogeneic stem cell transplantation, in which stem cells from a donor that is genetically similar to and HLA-compatible with the recipient are transplanted into the subject. In some aspects of this disclosure, autologous stem cells are used for HSCT. In some aspects of this disclosure, allogeneic stem cells that are HLA-compatible with the subject are used for HSCT. In autologous HSCT, a sample containing stem cells is taken from the subject, stored, and later transplanted into the subject.

[0095] Because hematopoietic stem cells (HSCs) constitute a small portion of the total population of blood cells in a sample, it may be advantageous to increase the number of autologous or allogeneic HSCs before administering them to a subject for the treatment of cancer or infection. In some aspects of this disclosure, hematopoietic stem cells are collected and augmented before transplanting them into a subject for treatment. In some aspects of this disclosure, hematopoietic stem cells are collected, augmented, and selected from a sample before transplanting them into a subject for treatment. In some aspects, a sample containing hematopoietic stem cells is obtained and processed in vitro to increase the number of stem cells in the sample before administering the hematopoietic stem cells to a subject. In some aspects, a sample containing hematopoietic stem cells is obtained and processed in vitro to increase the percentage of stem cells in the sample before administering the hematopoietic stem cells to a subject.

[0096] In one embodiment, stem cells can be enriched in the material used for transplantation. In another embodiment, enrichment can be carried out by selectively stimulating the proliferation / increase of stem cells in other cells collected from the subject. In yet another embodiment, stem cells can be enriched by isolating stem cells from other cells collected from the subject. Such selection may be so-called positive selection or negative selection. In positive selection, stem cells are isolated based on markers known to be present on stem cells but not on other cells. In some embodiments, in positive selection, stem cells are isolated based on the markers CCR2+, CD34+ and / or lin-, thereby enriching HSCs with the positive markers. In negative selection, non-stem cells are identified and removed based on markers on such other cells, leaving stem cells. In some embodiments, in negative selection, stem cells are isolated based on the marker CCR2-. In negative selection, HSCs are treated ex vivo to remove CCR2- cells, thereby enriching HSCs with the positive markers CCR2+, CD34+ and / or lin- before administering HSCs to the subject. Such selection procedures are known to those skilled in the art and include, but are not limited to, flow cytometry analysis, microbead-based isolation, adhesion assays, and / or ligand-based selection. In some embodiments, ligand-based selection is based on the presence of a CCR2 ligand, e.g., CCL2. In some embodiments, enriched HSCs can be grown in vitro before administration to a subject. In some embodiments, enriched HSCs can be grown in vitro and then positively selected again for CCR2+, CD34+, and / or lin- before administration to a subject. In some embodiments, enriched HSCs can be grown in vitro and negatively selected for CCR2- cells, where the CCR2- cells can be removed again before administering the HSCs to a subject. In some embodiments, after removal of CCR2- cells, less than 20% of the starting population of CCR2-HSCs remains. In some embodiments, after removal of CCR2- cells, less than 15%, less than 10%, less than 5%, less than 2%, and even less than 1% of the starting population of CCR2-HSCs remain.In some embodiments, removing CCR2-cells before administering HSCs to a subject results in HSCs for administration that contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% or less of CCR2-HSCs. In some embodiments, after positive selection for CCR2+, CD34+, and / or lin- cells; after positive selection for CCR2+, CD34+, and / or lin- cells and proliferation of positively selected cells; or after positive selection for CCR2+, CD34+, and / or lin- cells, proliferation of positively selected cells, and a second positive selection for CCR2+, CD34+, and / or lin- cells, and prior to administration of HSCs, the HSCs for administration comprise at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% CCR2+, CD34+, and / or lin-HSCs.

[0097] Sources of hematopoietic stem cells as used herein include: myeloid-depleted cells (lin-), cKit+ purified lineage-negative myeloid-derived cells, Sca+ purified lineage-negative myeloid-derived cells, cKit+Sca+ purified myeloid-derived cells, mobilization from host bone marrow using G-CSF, mobilization from host bone marrow using AMD3100, prelixafor, or the molecule 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane], umbilical cord blood or umbilical cord blood-derived stem cells, human leukocyte antigen (HLA)-matched blood, mesenchymal stem cells derived from blood or bone marrow, hematopoietic stem cells differentiated from induced pluripotent stem cells, mobilized peripheral blood, peripheral blood, hematopoietic stem cell subsets including lin- cells purified with the CCR2+ marker, lineage-negative purified peripheral blood, or CD34+ enriched peripheral blood. In some aspects of this disclosure, the source of HSCs is bone marrow. In some aspects of this disclosure, the source of HSCs is autologous or allogeneic, and optionally, the source is bone marrow, peripheral blood, umbilical cord blood, umbilical cord blood stem cells, or induced pluripotent stem cells.

[0098] Hematopoietic stem cell mobilizers. In some aspects of this disclosure, hematopoietic stem cell mobilizers are administered to a subject. HSC mobilization refers to the mobilization of HSCs from the bone marrow of the subject to the peripheral blood of the subject. In this application, HSC mobilizers include: granulocyte colony-stimulating factor (G-CSF), pegylated G-CSF (pegylated G-CSF), lenoglaci, glycosylated forms of G-CSF, CXC motif chemokine 2 (CXCL2), CXC chemokine receptor type 4 (CXCR-4), or prelixafor.

[0099] Combination treatment or combination therapy. Combination treatment or therapy refers to two therapies performed in combination. Combination may be as a single dosage form, but more typically it is performed in separate doses using separate dosing regimens. In some embodiments, combination therapy may mean immune checkpoint inhibitor therapy combined with hematopoietic stem cell transplantation therapy and / or hematopoietic stem cell mobilization therapy. Immune checkpoint inhibitor therapy refers to treatment of a subject with a disease (e.g., cancer or infection) by administering one or more immune checkpoint inhibitors to the subject. Hematopoietic stem cell transplantation therapy refers to treatment of a subject with a disease (e.g., cancer or infection) by administering hematopoietic stem cells. This may be done in combination with the administration of hematopoietic stem cell mobilization agents to the subject. Hematopoietic stem cells may be proliferated, pre-selected based on markers, treated with cytokines, and / or administered with cytokines before administration to the subject, as disclosed herein. In some embodiments, the subject receives chemotherapy and / or radiotherapy, which are methods commonly known in the art, concurrently with the immune checkpoint inhibitor and hematopoietic stem cell transplantation combination therapy. In such an embodiment, hematopoietic stem cells may be administered to the subject after the completion of radiotherapy. In such an embodiment, hematopoietic stem cells may be administered to the subject after the completion of chemotherapy. In such an embodiment, hematopoietic stem cells may be administered to the subject within six weeks after the completion of chemotherapy or radiotherapy. In such an embodiment, hematopoietic stem cells may be administered 0, 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the completion of chemotherapy or radiotherapy. In such an embodiment, immune checkpoint inhibitors may be administered before, concurrently with, or after radiotherapy or chemotherapy. In such an embodiment, immune checkpoint inhibitors may be administered to the subject 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the completion of chemotherapy or radiotherapy.

[0100] Monotherapy. Monotherapy refers to treatment with one or more immune checkpoint inhibitors, without hematopoietic stem cell transplantation and / or hematopoietic stem cell mobilization.

[0101] Subject. "Subject" means mammals, such as humans, non-human primates, dogs, cats, sheep, horses, cattle, pigs, mice, rats, rodents, or goats. In important embodiments, the subject and / or mammal is human.

[0102] Treatment. "To treat," "to treat," "treatment," and "therapy" include actions that occur while a subject is suffering from a condition that reduce the severity of the condition (or symptoms associated with the condition) or delay or slow the progression of the condition (or symptoms associated with the condition). This is a therapeutic treatment.

[0103] Effective dose. The subject is treated with an effective dose of the solution of this disclosure. The “effective dose” of a drug generally means an amount sufficient to induce a desired biological response, i.e., to treat the condition. As will be understood by those skilled in the art, the effective dose of the drugs described herein may vary depending on the condition being treated, the mode of administration, and factors such as the subject’s age, body composition, and health.

[0104] For therapeutic purposes, an effective dose is a sufficient amount to provide a therapeutic benefit in the treatment of a condition or to reduce or eliminate one or more symptoms associated with the condition. This may include an amount that improves the overall therapy, reduces or avoids the cause of the symptoms or condition, or enhances the therapeutic effect of another therapeutic agent.

[0105] Generally, an effective dose is administered to enhance the immune response in a subject. In relation to a specific disease or condition, "enhancing the immune response" means preventing the onset, inhibiting the progression, reversing the onset, or otherwise reducing or mitigating one or more symptoms of the disease or condition, such as one or more symptoms of cancer or one or more symptoms of an infection. Furthermore, an effective dose may be an amount that slows, stops, or reverses the growth of cancer cells or infectious pathogens in a subject. An example effective dose of hematopoietic stem cells for injection is approximately 2 × 10⁶ per kg of the subject's body weight. 6This refers to individual cells. The exemplary effective dose of hematopoietic stem cells for injection may be greater than or less than this amount. Examples include approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 × 10⁶ cells. 6 Contains cells / kg.

[0106] The following are example effective doses of drugs used in this technology: Anti-immune checkpoint antibody: 0.01 mg / kg to 20 mg / kg every 1 to 4 weeks. In this embodiment, such administration continues as long as the cancer or infection persists. In this embodiment, administration may last, for example, up to 156 weeks.

[0107] Anti-PD-1 antibody: 0.01 mg / kg to 20 mg / kg every 1 to 4 weeks. In this embodiment, such administration continues as long as the cancer or infection persists. In this embodiment, administration may be for up to, for example, 156 weeks. In this embodiment, pembrolizumab may be administered at 10 mg / kg every 2 weeks, 10 mg / kg every 3 weeks, or 2 mg / kg every 3 weeks, for example, up to 96 weeks; nivolumab may be administered at 0.1 to 10 mg / kg every 2 weeks, for example, up to 96 weeks; pizilizumab may be administered at 0.1 to 10 mg / kg every week, 0.1 to 10 mg / kg every 2 weeks, or 0.1 to 10 mg / kg every 3 weeks, for example, up to 96 weeks. In this embodiment, MEDI-0680 may be administered once every 2 weeks for a maximum of 1 year. In this embodiment, REGN2810 may be administered once every 2 weeks. In this embodiment, AMP224 can be administered at a dose of 10 mg / kg once every two weeks.

[0108] Anti-PD-L1 antibody: 0.01 mg / kg to 20 mg / kg is administered to the subject every 1 to 4 weeks. In embodiments, such administration continues as long as the cancer or infection persists. In embodiments, administration may be for, for example, up to 156 weeks. In embodiments, BMS-936559 / MDX-1105 may be administered, for example, at 1, 3, or 10 mg / kg every 2 weeks for up to 2 years; MPDL3280A / RG7446 may be administered, for example, at 1200 mg every 3 weeks for up to 1 year, or up to 2 years, or until disease progression; MSB0010718C / avelumab may be administered, for example, at 10 mg / kg once every 2 weeks until disease progression; and MEDI4736 may be administered, for example, every 1, 2, 3, or 4 weeks for up to 1 year, or up to 2 years. The effective doses of some drugs are currently being tested in clinical trials and may vary accordingly.

[0109] Anti-CTLA-4 antibody: 0.01 mg / kg to 20 mg / kg every 1 to 4 weeks. In this embodiment, such administration continues as long as the cancer or infection persists. In this embodiment, administration may be for, for example, up to 156 weeks. In this embodiment, MDX-010 / ipilimumab may be administered at 0.3 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg every 3 weeks for 4 doses or 4 cycles, or up to 32 doses, along with maintenance therapy every 12 weeks. Tremelimumab / CP-675,206 may be administered at 3 mg / kg, 6 mg / kg, 10 mg / kg, or 15 mg / kg every 12 weeks for 4 doses, or up to 8 doses. The effective doses of some drugs are currently being tested in clinical trials and may change accordingly.

[0110] Anti-VISTA antibody: 0.01 mg / kg to 20 mg / kg is administered to the patient every 1 to 4 weeks. In this embodiment, such administration continues for as long as the cancer or infection persists. In this embodiment, administration may continue for, for example, up to 156 weeks. The effective dose of some drugs may change depending on the results of future clinical trials.

[0111] Mobilizers: Such agents are administered in amounts sufficient to mobilize stem cells from the bone marrow into the peripheral blood. Such amounts for a particular mobilizer are, for example, 1 μg / kg to 20 μg / kg of G-CSF per day, preferably 5 μg / kg or 10 μg / kg of G-CSF per day; 1 to 20 mg of PEGylated G-CSF, preferably 6 mg or 12 mg of PEGylated G-CSF per day; 1 to 20 μg / kg of PEGylated G-CSF per day; 1 to 20 μg / kg of renoglutinism per day; 1 to 40 μg / m³ per day. 2 CXC chemokine receptor type 4 (CXCR-4); 1-40 μg / m² 2 This is a daily prelixafor.

[0112] Method of Administration. In some aspects of this disclosure, immune checkpoint inhibitors are administered intravenously (intravenous (IV) infusion). Antibodies may also be administered via other modes of administration known in the art. Such modes of administration include inhalation, ingestion, and topical application. Oral administration is also possible for therapeutic purposes, however this form of administration is more difficult for certain biological agents such as antibodies. HSCTs are often administered in conjunction with chemotherapy, which can be administered in a variety of ways. In aspects of this disclosure, HSC mobilizers are administered orally, subcutaneously, intramuscularly, intravenously, intraventricularly, intrathecally, intraperitoneally, intraarterially, intravesically, or intrapleurally, preferably intravenously.

[0113] Adoptive cell therapy (ACT or adoptive cell transfer). Adoptive cell therapy is the transfer of cells into a patient for the purpose of transmitting immune function and other properties along with the cells. The cells are most commonly immune-derived, e.g., T cells, and can be autologous or allogeneic. Transferring autologous cells rather than allogeneic cells minimizes the problem of graft-versus-host disease. ACT can be used to treat viral infections and / or to mitigate cancer regression. In subjects undergoing immunosuppressive or ablation procedures (e.g., chemical or radiological procedures), the risk of infection and / or malignancy is increased, for example, in association with HSCT, organ transplantation, and stem cell transplantation, including certain types of cancer (in which immune rearrangement is often slow and incomplete, and there is a risk of malignancy). The use of ACT in subjects during the post-immunosuppression period is considered beneficial to the subject due to the potential to enhance immunity, including anti-tumor immunity, and to increase vaccine efficacy during the post-immunosuppression period. ACT of tumor-specific T cells has been shown to be effective in treating mouse and human solid tumors. In one embodiment, ACT is used in conjunction with HSC infusion or administration of an HSC mobilizer, where the addition of HSCs increases the target's immune response, as indicated by increased IFNγ secretion. [Examples]

[0114] Example 1. Hematopoietic stem cells (HSCs) alter the tumor influx lymph node microenvironment and enhance anti-tumor immunity. HSC transfer leads to an increase in anti-tumor T cells that secrete IFNγ in tumor-hospitalizing hosts. Adoptive cell therapy (ACT), consisting of intravenous infusion of tumor-specific T cells and intradermal vaccination with dendritic cells, was administered to intracranial tumor-bearing mice, with or without intravenous infusion of hematopoietic stem cells (HSCs). The T cells were derived from mice possessing yellow fluorescent protein (YFP) under the control of the interferon-γ promoter, and therefore fluoresce upon activation. Lymph nodes in the tumor inflow area (cervical nodes) were collected from tumor-bearing mice after ACT+HSC, RNA was extracted, and the expression of a panel of T cell activation markers was examined by PCR array. Relative gene expression in the +HSC lymph nodes is shown as a heatmap in Figure 1A. A significant increase in interferon-γ is indicated by a red square (D6, seen in the color version of Figure 1A), which indicates IFNγ expression. T cell flow cytometry was used to examine YFP (IFNγ) expression (shown on the x-axis) in Figure 1B. Naive mice expressed less than 1% IFNγ, but tumor-specific T cells augmented in vitro showed approximately 3.5% cellular reactivity. After ACT without HSCs, the activated state increased to ~64.3% of IFNγ-secreting T cells. Addition of HSCs significantly enhanced IFNγ secretion, with >90% positivity in the presence of HSCs. The calculation of IFNγ+ T cells in each group is shown in Figure 1C.

[0115] Adoptive cell therapy: C57BL / 6 mice (Jackson Laboratories) have 10 4 5 × 10¹ KR158B astrocytoma cells were stereotactically transplanted into the right caudate nucleus on day 0. The mice were then administered a single dose of non-myeloablative (NMA) 5 Gy or myeloablative (MA) 9 Gy total body irradiation (TBI) on day 4. 5 × 10¹⁶ hematopoietic stem cell transplanted mice were then given 5 × 10¹⁶ 4 Lin-bone marrow-derived stem cells were intravenously injected within 24 hours of TBI. 10 7 Intravenous injection of tumor-specific T lymphocytes was administered between 16 and 24 hours after TBI. Immediately thereafter, 2.5 × 10⁶ 5 Intradermal vaccination was performed using all tumor RNA pulsed DCs. DC vaccines 2 and 3 were administered at weekly intervals.

[0116] Hematopoietic stem cell (HSC) isolation. Bone marrow was collected from the femur and tibia of C57BL / 6 mice. Red blood cells were then lysed using an ammonium chloride-based lysis solution (PharmLyse, BD Biosciences) to leave mononuclear cells. These cells were isolated using a Miltenyi Biotec mouse strain (lin-) removal kit according to the manufacturer's instructions. The cells were labeled with a Miltenyi biotin-labeled antibody cocktail, followed by labeling with a bead-conjugated secondary antibody. This solution was then passed through a sterile magnetic column to isolate lin- hematopoietic stem cells (HSCs). The HSCs were resuspended in sterile phosphate-buffered saline and intravenously injected into mice within 2 hours of isolation. Treated mice yielded 50,000–100,000 HSCs in a final volume of 100 μl.

[0117] Generation of tumor-specific T cells for adoptive transfer. Total RNA was isolated from KR158B-luc tumors and electroporated into bone marrow-derived dendritic cells (DCs) using the BTX Single Waveform Electroporation System (Harvard Apparatus). Naive mice were intradermally vaccinated with total tumor RNA-pulsed DCs, and spleens were harvested 7 days later. Splenocytes were augmented ex vivo for 7 days using RNA-pulsed DCs and 100 IU of IL-2 (R&D Systems). T cells were augmented from the spleens of either wild-type C57BL / 6 mice (Jackson Laboratories, Bar Harbor, ME, stock #000664), DsRed transgenic mice in a C57BL / 6 background (Jackson Laboratories, stock #006051), or GFP transgenic mice in a C57BL / 6 background (Jackson Laboratories, stock #004353) that received initial stimulation. Tumor-responsive T cells were intravenously adopted 5–7 days after in vitro activation.

[0118] Generation of RNA-pulsed dendritic cell (DC) vaccine. Dendritic cells (DCs) were isolated from the bone marrow of C57BL / 6 mice using a modified version of a previously published protocol. Briefly, the femurs and tibias of C57BL / 6 mice were harvested, and the bone marrow was washed with RPMI (LifeTechnologies) + 10% FBS (LifeTechnologies). Red blood cells were lysed in 10 mL of Pharmlyse (BD Bioscience), and mononuclear cells were resuspended in CDCM (RPMI-1640, 5% FBS, 1 M HEPES (LifeTechnologies), 50 μM and 55 mM β-mercaptoethanol (LifeTechnologies), 100 mM sodium pyruvate (LifeTechnologies), 10 mM non-essential amino acids (LifeTechnologies), 200 mM L-glutamine (LifeTechnologies), 10 μg GM-CSF (R&D Systems), 10 μg IL-4 (R&D Systems), and 5.5 mL penicillin / streptomycin (LifeTechnologies)). This was then transferred to a 6-well plate treated with tissue culture. 6 Cells were seeded at a density of cells / mL and a total volume of 3mL / well. Non-adherent cells were discarded on day 3. Non-adherent cells were collected on day 7 and placed on a 100mm tissue processing dish. 6 Cells were re-seed at a density of cells / mL and a total volume of 5mL / dish. After 24 hours, the obtained cells were electroporated with 25 μg of total RNA isolated from KR158B-luc cells (RNeasy, Qiagen). RNA-pulsed DCs were collected the following day and stored in PBS at a final concentration of 2.5 × 10⁶. 6 The cells were suspended, and 100 μl of the cell suspension was administered by intradermal injection.

[0119] Figure 1A shows that 7-week-old female C57BL / 6 mice were intracranially injected with 10,000 astrocytoma cells into the intracranial nucleus. On day 4, all mice received 5 Gy of non-myeloablative whole-body irradiation. Group 1 received tumor-specific T cell adoptive transfer and DC vaccine only. Group 2 received T cell adoptive transfer, DC vaccine, and HSC transfer. Lymph nodes were collected from both groups and RNA was isolated. PCR arrays were performed on RNA from the lymph nodes to examine genes associated with T cell activation. This figure shows the expression in the lymph nodes of mice that received HSC transfer compared to mice that did not. The results show a significant increase in IFNγ expression in the group that received HSC.

[0120] Figures 1B and 1C show the results of the same experiment as in Figure 1A, using T cells generated from YETI mice expressing yellow fluorescent protein (YFP) on the IFNγ promoter. Therefore, cells with antitumor function (IFNγ secretion) can be easily detected using flow cytometry. The spleens of these mice were analyzed for YFP, as well as newly generated tumor-specific T cells. YFP was hardly or never detected in splenic cells, and only 3-7% of the T cells augmented in vitro expressed YFP. These cells were then used for adoptive transfer into tumor-bearing mice under 5 Gy non-myeloablative host conditioning conditions. Group 1 received only T cells and DC vaccine, while Group 2 received HSCs, T cells, and DC vaccine. Lymph nodes in the tumor inflow area were resected and analyzed for YFP expression. Mice in Group 2 (T cells, DC vaccine, and HSC transfer) showed a significant increase in T cell activation. HSC transfer leads to an increase in anti-tumor T cells that secrete IFNγ in tumor-hospitalizing hosts. Figure 2 shows that the combination of HSCs and immune checkpoint inhibitors enhances the increase in IFNγ secretion by tumor-infiltrating host cells.

[0121] Example 2. Hematopoietic stem cells (HSCs) alter the tumor microenvironment and restore responsiveness to immune checkpoint blockade. The combination of HSCs and immune checkpoint inhibitors enhances increased IFNγ secretion by tumor-infiltrating host cells. The observation that HSCs alter the lymph node microenvironment and allow for increased T cell activation, as indicated by IFNγ production, prompted us to test the effects of HSC transfer on the tumor microenvironment and immune cell activation state within intracranial tumors. The tumor microenvironment is known to be significantly immunosuppressive, leading to the arrest of anti-tumor immune cells and the failure of immunological tumor rejection. We showed that HSC transfer alone resulted in an increase in intratumoral IFNγ-secreting cells, as shown in Figures 2A and 2B. This increase was greater than that induced by PD1 blockade with the anti-PD-1 monoclonal antibody. However, HSC transfer alone or anti-PD-1 monoclonal antibody treatment alone did not significantly extend survival in tumor-carrying animals (Figure 2C). However, the combination of HSC + anti-PD-1 antibody showed a remarkable synergistic effect in intratumoral immune activation, as indicated by IFNγ secretion (Figure 2B), resulting in significantly improved survival and long-term cure in >40% of treated animals (Figure 2C). These results demonstrate a novel role of HSCs in altering the tumor microenvironment and enhancing antitumor immunity in animals treated with anti-PD-1 antibodies. These effects enable refractory tumors to become sensitive to anti-PD-1 antibody treatment. The results of combining anti-PD-1 monoclonal antibody treatment that induces immune checkpoint blockade with HSC administration suggest to the inventors that other immune checkpoint inhibitors, such as CTLA-4 targeting antagonists, may be useful in combination therapy with HSCs. The synergistic effects of combining anti-PD-1 monoclonal antibody treatment that induces immune checkpoint blockade with HSC administration, as disclosed herein, suggest that antagonists targeting other immune checkpoints, such as PD-L1, CTLA-4, and / or VISTA, may be useful in combination therapy with HSCs.

[0122] Combining HSC therapy with treatment with a CTLA-4 antagonist did not yield conclusive results (data not shown). However, results obtained with combination therapy of HSC transfer and PD-1 or VISTA antagonism suggest that other immune checkpoint antagonists may produce synergistic effects when combined with HSC transfer. Combining treatment with multiple agents that antagonize one or more immune checkpoints with HSC transfer may produce synergistic effects. In some embodiments, one or more agents that antagonize one or more immune checkpoint molecules, e.g., PD-1, PD-L1, CTLA-4, and / or VISTA, are administered in combination with HSCT therapy. In some embodiments, synergistic effects may arise from administering one or more agents that antagonize one or more immune checkpoint molecules, e.g., PD-1, PD-L1, CTLA-4, and / or VISTA, in combination with HSC transfer and / or HSC mobilization treatment.

[0123] Example 3. The synergistic effect of combination therapy with HSC transfer and immune checkpoint inhibitors is further enhanced by radiotherapy. Immune-responsive C57BL / 6 mice were given intracranial tumors and then divided into seven groups: Group 1: Tumor only; Group 2: Lineage-negative hematopoietic stem cells (HSCs); Group 3: Anti-PD1 antibody (αPD1); Group 4: HSCs + αPD1; Group 5: 500 rads of whole-body irradiation + HSCs; Group 6: 500 rads of whole-body irradiation + αPD1; and Group 7: 500 rads of whole-body irradiation + HSCs + αPD1. For whole-body irradiation, a single dose of 500 rads of X-ray irradiation was administered 4 days after tumor transplantation. For intravenous HSC administration, 100% of the HSC was administered in sterile saline solution. 5 A single dose of cells was administered as a final volume of 100 μl five days after tumor transplantation. For intraperitoneal αPD1, 10 mg / kg was administered every five days for a total of four doses, with the starting dose five days after tumor transplantation.

[0124] Figure 5 shows the experimental results: Median survival was significantly extended from 45 days in the tumor-only control group to 52 days in the animals that received HSC+αPD1 (p=0.0104). Median survival was also significantly extended in the group that received 500 rads of whole-body irradiation + HSC+αPD1 compared to the tumor-only control group (p=0.0028). Importantly, the group that received HSC+αPD1 showed a significant benefit in median survival compared to the group that received αPD1 alone (p=0.0237). By adding irradiation, tumor-bearing mice that received irradiation + HSC+αPD1 (86 days) had a significantly longer median survival than the group that received irradiation + αPD1 (45 days) (p=0.0018). There was no statistically significant difference in survival between the HSC+αPD1 group and the irradiation + HSC+αPD1 group (p=0.04393).

[0125] Example 4. Combining HSC treatment with immune checkpoint blockade using anti-VISTA antibodies results in increased survival in mice with anti-VISTA resistant tumors. Immune-responsive C57BL / 6 mice were given intracranial tumors and then divided into four groups: Group 1: Tumors only; Group 2: Lineage-negative hematopoietic stem cells (HSCs); Group 3: Antibodies against the V-domain Ig suppressor of T cell activation (VISTA) (αVISTA); and Group 4: HSCs + αVISTA. For intravenous HSC administration, 100% sterile saline solution was used. 5 A single dose of cells, with a final volume of 100 μl, was administered 5 days after tumor transplantation. For intraperitoneal αVISTA, a dose of 300 ug was administered every 3 days for a total of 4 doses.

[0126] Figure 6 shows the experimental results: median survival was significantly extended from 43 days in the tumor-only control group to 46 days in animals treated with HSC+αVISTA (p=0.0332). These results suggest that other immune checkpoint inhibitors, when combined with HSC therapy, can be targeted in subjects to treat diseases, such as cancer and / or infections, and increase survival.

[0127] Example 5: CCR2-positive (CCR2pos or CCR2+) HSCs enhance the effectiveness of HSC therapy combined with αPD1 treatment. Immunoreactive C57BL / 6 mice were given intracranial gliomas and divided into eight groups: Group 1: Tumors only; Group 2: Lineage-negative hematopoietic stem cells (HSCs); Group 3: HSCs that do not express CCR2 (CCR2neg HSCs); Group 4: HSCs that express CCR2 (CCR2pos HSCs); Group 5: αPD1; Group 6: αPD1+ HSCs; Group 7: αPD1+CCR2neg HSCs; and Group 8: αPD1+CCR2pos HSCs. Negative selection of lineage commitment markers (lineage-negative (Lin-)) and positive selection of CCR2 (CCR2pos) on HSCs were performed alone and in combination with αPD1 treatment. For CCR2+ HSC selection, bone marrow-derived cells were first isolated using a magnetic lineage-deficient kit (Miltenyi Biotec). The resulting HSCs were then stained with biotinylated anti-CCR2 antibody (Miltenyi Biotec). Next, an anti-biotin antibody conjugated onto magnetic beads was added, and then the cell suspension was passed through a magnetic column. The resulting cell fractions were CCR2neg HSCs and CCR2pos HSCs.

[0128] The results are shown in Figure 7: The median survival in the group receiving αPD1+CCR2pos HSCs was significantly increased compared to the group receiving HSC+αPD1 (p=0.0323). This was an unexpected finding, as the immune-enhancing effects of CCR2pos HSCs had not been previously described. The use of this specific subset of CCR2+HSCs may be more beneficial when combined with immune checkpoint blockade.

[0129] Example 6. HSC transfer combined with PD-1 blockade enhances lymphocyte function and maintains T cell activation in the tumor microenvironment - IFNγ production is maintained in the tumor microenvironment. Immune-responsive mice were given intracranial tumors and divided into four groups: Group 1: Tumor only; Group 2: Tumor + HSC; Group 3: Tumor + αPD-1; Group 4: Tumor + HSC + αPD-1. Mice used as tumor-carrying hosts express yellow fluorescent protein (YFP) when secreting interferon-gamma (IFNγ). For intravenous HSC administration, a single dose of 105 cells in sterile saline was administered in a final volume of 100 μl five days after tumor transplantation. For intraperitoneal αPD1, a total of four doses of 10 mg / kg were administered every five days, with the starting dose five days after tumor transplantation.

[0130] The results are shown in Figures 8A and 8B: Quantification of YFP / IFNγ+CD3+ lymphocytes in the tumor microenvironment by flow cytometry analysis (Figure 8A) (Figure 8B) in untreated mice and mice treated with either HSC, anti-PD1, or both HSC and anti-PD1. The results show a significant increase in IFNγ secretion by CD3+ T cells in the combination therapy-treated group (p-value = 0.001). This demonstrates that HSC+anti-PD1 treatment leads to an increased frequency of tumor-infiltrating lymphocytes with antitumor responsiveness.

[0131] Example 7. HSCs enhance lymphocyte function in the tumor microenvironment along with PD-1 blockade. Immune-responsive C57BL / 6 mice were given intracranial tumors and divided into four groups: Group 1: tumor only; Group 2: tumor + HSC; Group 3: tumor + αPD-1; and Group 4: tumor + HSC + αPD-1. For intravenous HSC administration, a single dose of 10⁵ cells in sterile saline was administered in a final volume of 100 μl 14 days after tumor transplantation. For intraperitoneal αPD1, a total of four doses of 10 mg / kg were administered every 5 days, with the starting dose 14 days after tumor transplantation. 35 days after tumor transplantation, the tumors were harvested and RNA was isolated using a commercially available kit (Qiagen). RNA from the samples was analyzed using a T-cell and B-cell activated RT2 PCR array (SA Biosciences) according to the manufacturer's instructions.

[0132] Genetic analysis of tumors was performed from mice treated with HSC alone, anti-PD1 alone, or both HSC and anti-PD1. The results are shown in Figure 9: the enlarged portion of the gene expression heatmap shows that numerous genes involved in the T cell activation / inflammatory pathway, such as Fas1, IFNγ, and TNF, are highly upregulated in group 4 (tumor + HSC + αPD-1 treatment). The results demonstrate that the combined HSC + anti-PD1 treatment increases markers associated with activated cytotoxic T cells, including IFNγ. We also found upregulation of chemokines, known mediators of T cell migration.

Claims

1. A composition for use in increasing the immune capacity of a subject having an infectious disease, wherein the subject has undergone concurrent immune checkpoint inhibitor treatment with one or more immune checkpoint inhibitors, the composition comprises hematopoietic stem cells, each of which is an antagonist of programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or the V-domain Ig suppressor of T cell activation (VISTA), wherein the PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist, and VISTA antagonist are antibodies, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNAs, or siRNAs that selectively bind to any one of PD-1, PD-L1, PD-L2, and VISTA.

2. The composition according to claim 1, wherein hematopoietic stem cells are enriched with respect to CCR2+, CD34+, and / or lin- cells.

3. The composition according to claim 1, wherein the hematopoietic stem cells are substantially free of CCR2- cells.

4. The composition according to any one of claims 1 to 3, wherein the infection is resistant to monotherapy treatment with one or more immune checkpoint inhibitors.

5. A composition for use in increasing the immune capacity of a subject having an infectious disease, wherein the subject has received concurrent treatment with hematopoietic stem cells, preferably the infectious disease is resistant to monotherapy with an immune checkpoint inhibitor, and the composition comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antagonist of programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or the V-domain Ig suppressor of T cell activation (VISTA), and the PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist, and VISTA antagonist are antibodies, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNAs, or siRNAs that selectively bind to any one of PD-1, PD-L1, PD-L2, and VISTA.

6. A composition for use in increasing the immune capacity of a subject with an infectious disease, wherein the subject has undergone hematopoietic stem cell transplantation and / or concurrent treatment with a hematopoietic stem cell mobilizer, preferably the infectious disease is resistant to monotherapy with an immune checkpoint inhibitor, and the composition comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or T cell activation V domain Ig The composition is an antagonist of a suppressor (VISTA), wherein the PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist, and VISTA antagonist are antibodies, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNAs, or siRNAs that selectively bind to any one of PD-1, PD-L1, PD-L2, and VISTA.

7. The composition according to any one of claims 1 to 6, wherein the immune checkpoint inhibitor is a PD-1 antagonist, and the PD-1 antagonist is a humanized antibody that selectively binds to PD-1, preferably the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, pizilizumab, MEDI-0680, REGN2810, or AMP-224, or the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, or pizilizumab.

8. The composition according to any one of claims 1 to 6, wherein the immune checkpoint inhibitor is a PD-L1 antagonist, and the PD-L1 antagonist is a humanized antibody that selectively binds to PD-L1, preferably the humanized antibody that selectively binds to PD-L1 is BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab.

9. The composition according to any one of claims 1 to 6, wherein the immune checkpoint inhibitor is a VISTA antagonist, and the VISTA antagonist is an inhibitory antibody against VISTA.

10. The composition according to any one of claims 1 to 4 and 7 to 9, wherein the immune checkpoint inhibitor is administered on a different day from the hematopoietic stem cells, or the immune checkpoint inhibitor is administered on the same day as the hematopoietic stem cells, or the immune checkpoint inhibitor is administered on a different day from the hematopoietic stem cells, but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months of the hematopoietic stem cells.

11. The composition according to any one of claims 5 and 7 to 9, wherein the hematopoietic stem cells are administered on a different day from the immune checkpoint inhibitor, or the hematopoietic stem cells are administered on the same day as the immune checkpoint inhibitor, or the hematopoietic stem cells are administered on a different day from the immune checkpoint inhibitor, but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months of the immune checkpoint inhibitor.

12. The composition according to any one of claims 6 to 9, wherein the hematopoietic stem cell transplant and / or hematopoietic stem cell mobilizer is administered on a different day from the immune checkpoint inhibitor, or the hematopoietic stem cell transplant and / or hematopoietic stem cell mobilizer is administered on the same day as the immune checkpoint inhibitor, or the hematopoietic stem cell transplant and / or hematopoietic stem cell mobilizer is administered on a different day from the immune checkpoint inhibitor, but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months of the immune checkpoint inhibitor.

13. The composition according to any one of claims 1 to 12, further comprising administering a hematopoietic stem cell mobilizing agent to the subject.

14. The composition according to any one of claims 1 to 13, wherein the infection is a chronic infection, or the infection is any hepatitis, adenovirus, polyomavirus such as BK, human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B and / or C, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), staphylococcal species including methicillin-resistant Staphylococcus aureus (MRSA), streptococcal species including Streptococcus pneumoniae, or a post-transplant infection, preferably the infection is hepatitis A, hepatitis B, or hepatitis C.

15. The source of hematopoietic stem cells is bone marrow, myeloid-depleted cells (lin-), cKit+ purified lineage-negative bone marrow-derived cells, Sca+ purified lineage-negative bone marrow-derived cells, cKit+Sca+ purified bone marrow-derived cells, GM-CSF, G-CSF, mobilization from host bone marrow using AMD3100, prelixafor, or the molecule 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane], umbilical cord blood or umbilical cord blood-derived stem cells, human leukocyte antigen (HLA) compatible blood, blood or bone The composition according to any one of claims 1 to 14, wherein the source of hematopoietic stem cells is bone marrow-derived mesenchymal stem cells, hematopoietic stem cells differentiated from induced pluripotent stem cells, mobilized peripheral blood, peripheral blood, a hematopoietic stem cell subset including lin- cells purified with a CCR2+ marker, lineage-negative purified peripheral blood, or CD34+ enriched peripheral blood, or the source of hematopoietic stem cells is bone marrow, peripheral blood, umbilical cord blood, or induced pluripotent stem cells, or the source of hematopoietic stem cells is autologous, or the source of hematopoietic stem cells is allogeneic and the donor cells are HLA-compatible with the recipient.

16. The composition according to any one of claims 1 to 15, wherein the effect of increasing the immune capacity of the target is evaluated by measuring the secretion of interferon-gamma (IFNγ) by T cells obtained from the tumor microenvironment or tumor inflow area lymph nodes of the target, and a synergistic effect is observed when the presence of IFNγ increases with combination therapy.

17. The composition according to any one of claims 1 to 16, wherein at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 percent of the hematopoietic stem cells are CCR2-positive (CCR2+), CD34-positive (CCR2+), and / or lineage-negative (lin-) cells.

18. The composition according to any one of claims 1 to 16, wherein hematopoietic stem cells for administration to a subject are enriched ex vivo with CCR2-positive (CCR2+) cells, CD34-positive (CD34+) cells, and / or lineage-negative (lin-) cells before administration to the subject, or the hematopoietic stem cells are treated ex vivo before administration to the subject to remove CCR2-negative (CCR2-) cells, or the hematopoietic stem cells are selected for CCR2+, CD34+, and / or lin- cells before administration to the subject by flow cytometry analysis, microbead-based isolation, adhesion assay, and / or ligand-based selection, preferably the cells are selected by ligand-based selection, and the ligand is a CCR2 ligand known as CCL2.

19. A composition for use in increasing the immune capacity of a subject with an infectious disease, wherein the increase in the subject's immune capacity is To administer stem cell mobilization agents as the target, To collect hematopoietic stem cells from the subject, The collected stem cells are enriched with CCR2-positive (CCR2+), CD34-positive (CD34+), or lineage-negative (lin-) cells. Optionally, remove the collected stem cells or CCR2- cells. The subjects are administered enriched collected stem cells, and The target group will be given immune checkpoint inhibitors. The composition comprises hematopoietic stem cells, wherein the immune checkpoint inhibitor is an antagonist of programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or V-domain Ig suppressor for T cell activation (VISTA), and the PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist, and VISTA antagonist are antibodies, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNAs, or siRNAs that selectively bind to any one of PD-1, PD-L1, PD-L2, and VISTA.

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