Compositions and methods for activating immune cells

Abstract

The present application provides compositions and methods for producing antigen-presenting cells (APCs) from monocytes (e.g., monocytes from cancer patients) comprising an IL-10 receptor activator (e.g., IL-10), an IFNγ receptor activator (e.g., IFNγ), a TNFα receptor activator (e.g., TNFα), an IL-4 receptor activator (e.g., IL-4), a GM-CSF receptor activator (e.g., GM-CSF), and / or an IL-6 receptor activator (e.g., IL-6). APCs produced accordingly, as well as methods for activating immune cells (e.g., T cells) via co-culture with APCs, are also provided. Activated immune cell compositions and treatment methods comprising the activated immune cells are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,439, filed March 30, 2022, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] FIELD OF THEINVENTION The present invention relates to compositions and methods for a) promoting survival of monocytes, b) promoting differentiation and / or maturation of antigen-presenting cells (APCs) derived from monocytes (e.g., monocytes from cancer patients or healthy donors), and c) activating immune cells (e.g., T cells). [Background technology]

[0003] 2. Background of the Invention Professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, and their mediated network of immunogenic immunity, are central components for establishing antigen-specific adaptive immunity that protects the host from cancer mutations, infectious pathogens and injury. In cancer immunotherapy, the promise of APCs is their ability to phagocytose tumor cells and then present antigens that activate tumor-specific adaptive immunity, including tumoricidal T cells and long-lasting anti-cancer antibodies. In addition, successful production of APCs will also facilitate the development of APC vaccines to treat infectious diseases, such as those caused by viruses, bacteria and other pathogens. With these capabilities, APC-based therapy, once established, is expected to achieve complete cancer curative efficacy, systemically eliminating tumors and metastases and establishing immune memory to prevent recurrence / relapse.

[0004] However, the use of APCs for cancer treatment over the years has revealed some significant limitations. For example, the isolation and ex vivo stimulation of autologous APCs is time-consuming and expensive, and the quality of ex vivo-generated DCs has been found to be variable. Thus, the use of patient-derived autologous DCs limits the standardization of DC-based treatment protocols. See, for example, Eggermont et al., Trends Biotechnol. 2014 Sep;32(9):456-65. Among these, one of the most important barriers to APC therapy is the lack of technology to reliably drive monocytes from cancer patients to differentiate into effective proinflammatory DC / macrophage-like APCs. The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Eggermont et al., Trends Biotechnol.2014 Sep;32(9):456-65 Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The application provides, in one aspect, a method of stimulating a population of monocytes from an individual to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs. In some embodiments, the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). See, e.g., Yang et al., Cytokine Growth Factor Rev. 2019 Oct;49:10-22. In some embodiments, the IL-10R activator is IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml).

[0007] In some embodiments according to any of the above methods, the plurality of S / D / M factors comprises an IL-4R activator, optionally the IL-4R activator is selected from the group consisting of IL-4, IL-13, an IL-4R agonist antibody, and a small molecule activator of IL-4R. In some embodiments, the IL-4R activator is IL-4. In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4R activator is IL-13. In some embodiments, the IL-13 is human IL-13 or human recombinant IL-13. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml). In some embodiments, IL-13 is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 60 pg / ml, and further optionally from about 60 pg / ml to about 2 ng / ml (e.g., from about 100 pg / ml to about 2 ng / ml).

[0008] In some embodiments according to any of the methods described above, the plurality of S / D / M factors comprises a TNFR activator, optionally the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml).

[0009] In some embodiments according to any of the methods described above, the plurality of S / D / M factors comprises an IFNGR activator, optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR. In some embodiments, the IFNGR activator is IFNγ. In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml).

[0010] In some embodiments according to any of the above methods, the multiple S / D / M factors are present in a single composition.

[0011] In some embodiments according to any of the methods described above, at least one of the plurality of S / D / M factors is provided separately from one of the other S / D / M factors of the plurality of S / D / M factors.

[0012] In some embodiments according to any of the methods described above, the plurality of S / D / M factors comprises two or more agents selected from the group consisting of IL-4R activators, TNFR activators, and IFNGR activators, hi some embodiments, the plurality of S / D / M factors comprises IL-10, IL-4, TNFα, and IFNγ.

[0013] In some embodiments according to any of the methods described above, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator. In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml).

[0014] In some embodiments according to any of the methods described above, the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, optionally the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R. In some embodiments, the IL-6R activator is IL-6. In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally from about 5 pg / ml to about 100 pg / ml (e.g., from about 10 to 50 pg / ml, e.g., about 30 pg / ml).

[0015] In some embodiments according to any of the methods described above, the plurality of S / D / M factors is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies, and optionally the plurality of S / D / M factors is derived from a supernatant of the culture. In some embodiments, the T cells are isolated from PBMCs of the same individual or a different individual. In some embodiments, the T cells are CD4 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the T cells have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the T cells have been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the plurality of S / D / M factors is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally for about 2 days.

[0016] In some embodiments according to any of the methods described above, the monocytes are cultured in the presence of S / D / M factors or medium derived from a culture of T cells for at least about 2 days (e.g., about 2-4 days, about 2-3 days, about 2 days).

[0017] In some embodiments of any of the methods described above, the method further comprises contacting the population of monocytes with a plurality of expansion refinement factors selected from the group consisting of a type I interferon, IFNγ, TNFα, a TLR ligand, CD40L or CD40 ligating antibody, an anti-PD-L1 antibody, and TPI-1, optionally wherein the type I interferon comprises IFNα and / or IFNβ, and optionally wherein the TLR ligand is Poly IC, CpG, or LPS. In some embodiments, the plurality of refinement factors is provided after contacting the plurality of monocytes with a plurality of S / D / M factors or medium derived from a culture of T cells, thereby producing a population of APCs, and the population of APCs is cultured in the presence of the plurality of refinement factors for about 1-5 days, and optionally wherein the population of APCs is cultured for about 1 day. In some embodiments, the refinement factors are provided when a) at least about 50% of the monocytes are viable, b) at least about 30% of the population of APCs exhibit dendritic cell morphology, and / or c) the population of APCs express i) high levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86, and / or CD40, and / or ii) low levels of SIRPα. In some embodiments, the refinement factors include IFNα, IFNγ, and TNFα. In some embodiments, the refinement factors further include Poly IC, CpG, CD40L, and an anti-PD-L1 antibody.

[0018] In another aspect, the application provides a method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium having one or more molecules that promote IL-10 receptor (IL-10R) expression on the monocytes. In some embodiments, the one or more molecules comprise an IL-10R activator, optionally wherein the IL-10R activator is selected from the group consisting of IL-10, an IL-10R agonist antibody, and a small molecule activator of IL-10R, and further optionally wherein the IL-10R activator is IL-10.

[0019] In another aspect, the application provides a method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium having an IL-10R activator, optionally wherein the IL-10R activator is selected from the group consisting of IL-10, an IL-10R agonist antibody and a small molecule activator of IL-10R, and further optionally wherein the IL-10R activator is IL-10.

[0020] In some embodiments according to the methods of promoting survival of a population of monocytes discussed above, the population of monocytes expresses low levels of IL-10R prior to contact with the molecule.

[0021] In some embodiments of the methods of promoting survival of a population of monocytes discussed above, the culture comprises a TNFα receptor (TNFR) activator and / or an interferon gamma (IFNγ) receptor (IFNGR) activator, optionally wherein the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, optionally wherein the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally wherein the culture comprises TNFα and / or IFNγ.

[0022] In another aspect, the application provides a method of increasing expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual with cancer, comprising contacting the population of monocytes with one or more agents selected from the group consisting of an IL-10R activator, a TNFR activator, and an IFNGR activator.

[0023] In another aspect, the application provides a method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium comprising IL-10, TNFα and IFNγ.

[0024] In another aspect, the application provides a method of promoting differentiation of a population of monocytes from individuals into antigen presenting cells ("APCs") in in vitro culture, comprising culturing the population of monocytes in a medium having one or more molecules selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator. In some embodiments, the culture further comprises an IL-6 receptor (IL-6R) activator and / or a GM-CSF receptor (GM-CSFR) activator.

[0025] In some embodiments according to any of the methods discussed above, the plurality of monocytes is obtained from the peripheral blood of the individual, and optionally, the monocytes express CD14 and are obtained from the peripheral blood.

[0026] In some embodiments according to any of the methods discussed above, the individual has cancer. In some embodiments, the individual has late stage cancer. In some embodiments, the individual has a solid tumor.

[0027] In some embodiments according to any of the methods discussed above, the individual has an inoperable tumor and / or metastasis.

[0028] In some embodiments according to any of the methods discussed above, the individual is a human.

[0029] In another aspect, the present application provides a population of APCs produced by any of the methods for producing a population of APCs described above. In some embodiments, the APCs express low levels of an inhibitory signaling molecule, and the inhibitory signaling molecule is selected from the group consisting of TGFβR, SIRPα, LIlRB and Siglec 10.

[0030] In another aspect, the application provides a population of APCs, the APCs expressing high levels of one or more antigen presenting molecules, the antigen presenting molecules being selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL and CD40, rather than dendritic cells obtained from healthy humans and cultured with GM-CSF and IL-4 for about 5 days, and optionally the APCs are generated from monocytes in ex vivo cell culture, and further optionally the monocytes are obtained from a cancer patient. In some embodiments, the APCs express low levels of inhibitory signaling molecules, the inhibitory signaling molecules being selected from the group consisting of TGFβR, SIRPα, LIlRB and Siglec 10.

[0031] In another aspect, the present application provides a method of activating a population of immune cells comprising co-culturing the population of immune cells with a population of APCs of any one of the populations of APCs described above, the APCs being pre-loaded with one or more neo-antigenic peptides. In some embodiments, the method comprises contacting the APCs with a composition comprising a plurality of neo-antigenic peptides, and / or the APCs are pre-incubated with the composition. In some embodiments, the composition comprising a plurality of neo-antigenic peptides is a surgical resection of tumor tissue or a biopsy extract thereof. In some embodiments, the composition comprising a plurality of neo-antigenic peptides is a mixture of tumor cells or extracts thereof isolated from tumor tissue or a biopsy. In some embodiments, the composition comprising a plurality of neo-antigenic peptides is a mixture of isolated neo-antigenic peptides. In some embodiments, the isolated neo-antigenic peptides are synthetic peptides. In some embodiments, the APCs are contacted with the composition comprising a plurality of neo-antigenic peptides for about 4 to about 24 hours. In some embodiments, the immune cells are selected from the group consisting of PBMCs, tumor infiltrating T cells (TILs), and T cells, and optionally the T cells are CD8 T cells and / or CD4 T cells. In some embodiments, the co-culturing is performed for at least 24 hours. In some embodiments, the method further comprises expanding the population of immune cells after the co-culturing step. In some embodiments, expanding the population of immune cells comprises contacting the immune cells with a cytokine selected from the group consisting of IL-2, IL-7 and IL-15, optionally for about 2 days to about 10 days. In some embodiments, the population of immune cells and the antigen-presenting cells are derived from the same individual. In some embodiments, the population of immune cells and the antigen-presenting cells are not derived from the same individual.

[0032] In another aspect, the application provides a population of activated immune cells obtained by any of the above methods of activating a population of immune cells.

[0033] In another aspect, the present application provides a method of treating cancer in a patient, comprising administering to the patient APCs and / or a population of activated immune cells according to any of the above populations of APCs and / or activated immune cells. In some embodiments, the APCs or activated immune cells are administered intratumorally, intraperitoneally, or intravenously. In some embodiments, the activated immune cells are administered in a dose of about 10 7 ~10 9 In some embodiments, the method further comprises treating the patient with chemotherapy, radiation therapy, or an immune checkpoint inhibitor. In some embodiments, the method comprises treating the patient with irradiation. In some embodiments, the irradiation site is different from the site of the cancer being treated. In some embodiments, the APC or activated immune cells administered to the patient are derived from the patient. In some embodiments, the APC or activated immune cells administered to the patient are not derived from the patient. In some embodiments, the cancer being treated is a solid tumor.

[0034] In another aspect, the application provides a composition comprising a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) an IL-10 receptor (IL-10R) activator, and 2) one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator. In some embodiments, the IL-10R activator is selected from the group consisting of IL-10, an IL-10R agonist antibody, and a small molecule activator of IL-10R. In some embodiments, the IL-10R activator is IL-10. In some embodiments, the plurality of S / D / M factors comprises an IL-4R activator, optionally the IL-4R activator is selected from the group consisting of IL-4, IL-13, an IL-4R agonist antibody, and a small molecule activator of IL-4R. In some embodiments, the IL-4R activator is IL-4. In some embodiments, the plurality of S / D / M factors comprises a TNFR activator, optionally the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the plurality of S / D / M factors comprises an IFNGR activator, optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR. In some embodiments, the IFNGR activator is IFNγ. In some embodiments, the plurality of S / D / M factors comprises two or more agents selected from the group consisting of an IL-4R activator, a TNFR activator, and an IFNGR activator. In some embodiments, the plurality of S / D / M factors comprises IL-10, IL-4, TNFα, and IFNγ. In some embodiments, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, optionally, the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R. In some embodiments, the IL-6R activator is IL-6. [Brief description of the drawings]

[0035] [Figure 1-1] Figures 1A-1E show the failure of monocytes from cancer patients (cMo) to respond to the macrophage / DC differentiation factors M-CSF and GM-CSF to induce differentiation of monocytes from healthy donors (Mo). Figures 1A-1C show that a high percentage of cMo cell death was associated with failure to differentiate. Figure 1C shows that a combination of tumor-conditioned media partially protected cMo from cell death but failed to induce differentiation with M-CSF plus GM-CSF. Figure 1D shows cMo showing reduced MCSF-R and GMCSF-R expression compared to Mo. Figure 1E shows that a combination of various reagents failed to support cMo survival or differentiation. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0036] [Figure 2-1]Figures 2A-C demonstrate the different signaling events occurring in Mo derived from healthy individuals and Mo derived from cancer patients in response to M-CSF and GM-CSF. Figure 2A shows that M-CSF and GM-CSF 1) caused activation of Akt and Erk1 / 2 in Mo but not cMo, and 2) caused caspase-9 and caspase-3 cleavage in cMo but not Mo, as demonstrated by increased phosphorylation of both Akt and Erk1 / 2 when assayed by Western blotting. Figure 2B shows a significant decrease in the percentage of cMo cell survival compared to the percentage of Mo cell survival as a result of apoptosis. Figure 2C shows the total levels of common intracellular signaling proteins, including Akt and Erk1 / 2, which were not decreased in cMo cells compared to Mo. [Figure 2-2] Same as above.

[0037] [Figure 3-1]3A-3I show the differentiation of monocytes from a cancer patient (cMo) with TCR-activated T cell conditioned medium (Karnelian X1 or Carnelian X1). FIG. 3A shows the experimental scheme. FIG. 3B-3C show the levels of cytokines produced from the first TCR stimulation of CD4 T cells derived from an individual with an autoimmune disorder, and the results demonstrated high levels of IL-10 production. FIG. 3D shows the cytokine profile of CD4 and CD8 T cells derived from an individual with an autoimmune disorder after the first and second TCR stimulations. FIG. 3E and FIG. 3F show the morphology of cMo from patient SD-21-451 under treatment with activated T cell medium or M-CSF. FIG. 3F shows that medium conditioned by TCR-activated T cells induces cMo differentiation. Figures 3G and 3H show the morphology and cell surface expression of antigen-presenting machinery of cMo from patient SD-21-451 after differentiation with Karnelian X1 for 72 hours, indicating that Karnelian X1 differentiates cMo into professional APCs. Figure 3I shows the percentage of cMo that survived and differentiated into κ APCs, which was strongly supported using medium with high IL-10 concentration (i.e., KX1 medium) but not healthy CD4 Te cell medium lacking IL-10. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0038] [Figure 4] FIG. 4 shows how monocytes from healthy donors or cancer patients survive and differentiate under different conditions (eg, M-CSF, GM-CSF or Karnelian X1).

[0039] [Figure 5-1]Figures 5A-5K show the key components of Karnelian X1. Figure 5A shows the cytokines produced by T cells after TCR stimulation. Media collected on day 2 (48 hours after stimulation) was referred to as Karnelian X1. Figure 5B shows that cytokine depletion assays identified IL-10, IFNγ and TNFα in Karnelian X1 as important for cMo survival. Figure 5C shows that IL-10 (recombinant) restores IL-10-depleted Karnelian X1 in a dose-dependent manner to support cMo survival. Figure 5D shows percent cMo survival when treated with JAK inhibitor, C188-9 or STAT3 inhibitor, napabucasin, in a dose-dependent reduction in cMo survival. Figure 5E shows that both STAT3 activators, colivelin TFA and garcinone D, restored cMo survival in a dose-dependent manner for cMo cells grown in media with low IL-10 concentrations. FIG. 5F shows percent cMo survival for cells cultured with media containing IL-10 family member cytokines (i.e., IL-19, IL-20, IL-22, and IL-24), which showed a similar dose-dependent response to treatment with IL-10, thereby restoring cMo survival. FIG. 5G shows percent cMo survival for cells cultured with media containing IL-12 family member cytokines (i.e., IL-12 and IL-23), which showed a similar pattern to treatment with IL-10, but at lower levels, thereby restoring cMo survival. FIG. 5H shows that IL-12 treatment induced the production of IL-10 by cMo. FIG. 5I provides a summary graphical representation of cMo survival and differentiation into κ APC in response to treatment with specific cytokines or STAT3 activators. FIG. 5J shows percent cMo survival when treated with IL-6 or IL-11 in low IL-10 media, which was significantly reduced compared to cMo cultured in media containing IL-10. FIG. 5K shows the percent of cMo survival when treated with G-CSF in low IL-10 medium, which is significantly reduced compared to cMo cultured in medium containing IL-10. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0040] [Figure 6-1] Figures 6A-G show the phenotype of cMo grown in media supplemented or lacking specific cytokines. Figure 6A shows that cytokine depletion assays identified that IL-4, IFNγ and TNFα were important for cMo phenotypic differentiation into APCs, and depletion of IFNγ, TNFα or IL-4 delayed cMo differentiation into APCs as indicated by reduced expression of MHC-II, CD80 / 86 and CD40, respectively. Figures 6B and 6D show that a cytokine cocktail (C-combo) recapitulated Karnelian X1 to support cMo survival and APC differentiation. Figure 6C shows the effect of KX1 treatment on cMo derived from patients with different stages of liquid or solid cancer. Figures 6E-G show a summary of the components of Karnelian X1 that support cMo differentiation into APCs over 1 and 2 days, respectively. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0041] [Figure 7-1] Figures 7A-7C show the mechanism by which Carnelian X1 induces cMo survival and differentiation into APCs. Figures 7A-7B show cytokine receptor expression on cMo before and after Carnelian X1 treatment compared to Mo from healthy donors. Figure 7C shows the hypothesis that cytokine-mediated upregulation of IL-10R leads to cMo survival and subsequent differentiation into immunogenic APCs with GM-CSF, TNFα, IL-6 and IFNγ-mediated phenotypes. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0042] [Figure 8-1]Figures 8A-C show that KX1 activates PI3K-Akt, MAPK and STAT3 cell survival pathways and avoids caspase-mediated apoptosis. Figure 8A shows that KX1 induced activation of Akt, Erk1 / 2 and STAT3 in cMo, as demonstrated by protein phosphorylation levels assessed by Western blotting, whereas both M-CSF and GM-CSF failed. Figure 8B demonstrates that KX1 treatment prevents cMo from undergoing apoptosis, as demonstrated by the levels of cleaved caspase-9 and caspase-3 measured by Western blotting. Figure 8C shows the involvement of PI3K-Akt and MAPK pathways in KX1-mediated regulation of cMo survival, such that inhibition of PI3K, Akt or MAPK reduces cMo survival, whereas inhibition of NFκB has no effect on cMo survival. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0043] [Figure 9-1] Figures 9A-9D show that IL-10 induced survival signals in cMo. Figures 9A-9B show cMo cultured with no treatment (NT), KX1, or KX1 supplemented with individual cytokine component depletion. Depletion of IL-10, IFNγ, and / or TNFα resulted in a decrease in cell survival and an increase in cleaved caspase-9 and caspase-3. Figure 9C shows the activation of Akt, Erk1 / 2, and STAT3 over time, as measured by protein phosphorylation levels, in cMo grown in KX1 with IL-10 or depleted of IL-10. Figure 9D shows the densitometry results calculated from the Western blotting images taken from Figure 9C. [Figure 9-2] Same as above.

[0044] [Figure 10-1]Figure 10A-D shows C-combo composition analysis. Figure 10A-C shows C-combo V1-V4 designed to test the minimal compositional requirements for activation of Akt, Erk1 / 2 and STAT3 in cMo (Figure 10A-B) and support cMo differentiation into κAPC (Figure 10C). Figure 10D compares KX1 medium rich in IL-10 with C-combo and finds similar activation of Akt, Erk1 / 2 and STAT3 in cMo. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0045] [Figure 11] FIG. 1 shows the phenotype of κ APCs differentiated from cMo, comparing the expression of antigen-presenting machinery in cells derived from cancer patients and grown in KX1 or C-combo medium with IL-10 supplementation.

[0046] [Figure 12-1]12A-12H show optimization of APC phenotype with Karnelian reagents (FIGS. 12A-12B: Opt1-4; FIGS. 12C-12D: Opt1-5). FIG. 12A shows the phenotype of APC differentiated from cMo derived from cancer patients after a) 48 hours of treatment with Karnelian X1, and b) an additional 24 hours of treatment with Opt1, 2, 3, or 4. Karnelian X2 is Opt3, as identified in FIG. 12B. FIG. 12C shows the phenotype of APC differentiated from cMo derived from cancer patients after a) 48 hours of treatment with Karnelian X1, and b) an additional 24 hours of treatment with Opt1, 2, 3, 4, or 5. FIG. 12D shows that Karnelian X2 is Opt5. FIG. 12E shows determination of pro-inflammatory κAPC phenotype by detecting inflammatory cytokines secreted by κAPC into cell-free medium. Figure 12F shows the determination of κAPC phagocytosis of tumor debris, CFSE-stained tumor cells were flash frozen in liquid N2, subsequently thawed on ice, and tumor cell debris was added to κAPC cultures in KX2 Opt5. Figure 12G shows the analysis of cell surface inhibitory receptors on κAPC, which showed low receptor levels except for increased expression identified for LILRB1 and LILRB3. Inhibition of SHP-1 by TPI-1 was used to enhance the proinflammatory response. Figure 12H shows an example of an enhanced proinflammatory response to TPI-1 treatment. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above.

[0047] [Figure 13-1] Figure 13A-B show the κAPC production protocol using KX1 and KX2. Figure 13A shows an overview of the production of κAPC from cMo with Karnelian X1 and X2 reagents. Karnelian X1 can be replaced with C-combo. Figure 13B shows the surface expression of various molecules on fresh cMo and cMo-derived APC after culture with Karnelian X1 and Karnelian X2. [Figure 13-2] Same as above.

[0048] [Figure 14-1] FIG. 14A shows the cell morphology of κAPCs assessed by light microscopy compared to immature DCs, mature DCs, M0 macrophages (MΦ), M1 MΦ, and M2 MΦ using KX1 and / or KX2, and FIG. 14B shows the size of κAPCs compared to other cell types. [Figure 14-2] Same as above. [Figure 14-3] Same as above.

[0049] [Figure 15-1] Figures 15A-B show the antigen-presenting capacity of cMo-derived κAPCs, comparing healthy Mo-derived κAPCs with MoDCs and M1 and M2 MΦs. Figure 15A shows a comparison of healthy Mo-derived κAPCs with MoDCs, M1 MΦs, and M2 MΦs from healthy individuals. Figure 15B shows the level of cMo-derived κAPC antigen-presenting capacity assessed by flow cytometry, where cMos were derived from patients with late-stage inoperable prostate, colorectal, or pancreatic cancer. [Figure 15-2] Same as above.

[0050] [Figure 16-1] Figures 16A-C show gene signatures from nanostring transcriptional profiling of Mo, cMo, Mo-derived κAPC, and cMo-derived κAPC. Figure 16A shows gene signatures of Mo or cMo compared to LPS-treated mature MoDC, LPS and IFNγ-treated MΦ, and κAPC derived from healthy individuals and cancer patients. Figure 16B shows transcriptional analysis of Mo, cMo, Mo-derived κAPC, and cMo-derived κAPC, demonstrating that Mo and cMo share similar gene signatures, but some differences were identified in Mo-derived κAPC and cMo-derived κAPC gene signatures. Figure 16C provides transcriptional analysis of cMo-derived κAPC grown in KX1 medium versus C-combo medium, in which gene signatures were similar. [Figure 16-2]Same as above. [Figure 16-3] Same as above.

[0051] [Figure 17-1] FIG. 17 shows pairwise comparisons of 1) gene transcription in Mo-derived κAPC vs. Mo, Mo-derived κAPC vs. M1 MΦ, and Mo-derived κAPC vs. LPS-stimulated MoDC; 2) cMo-derived κAPC vs. cMo and C-combo vs. KX1 medium; and 3) Mo vs. cMo and Mo-derived κAPC vs. cMo-derived κAPC. [Figure 17-2] Same as above.

[0052] [Figure 18-1] Figures 18A-B show the expression of different cell surface markers on κAPC compared to DC or MΦ APC. Figure 18A shows the cell surface markers found on different DC subtypes on Mo-derived DC (MoDC), M1 MΦ and κAPC. Figure 18B shows flow cytometry analysis of the cell surface markers identified in Figure 18A, demonstrating that κAPC does not share the same profile with cDC1, cDC2 or pDC, or with MoDC or M1 MΦ. [Figure 18-2] Same as above.

[0053] [Figure 19-1] Figure 19A-C provide identification of κAPC-specific cell surface markers. Figure 19A shows a comparison of cell surface expression between κAPC and MoDC and M1 MΦ, where cell surface proteins were specifically increased on κAPC (i.e., IL-3R and CD32; indicated by asterisks). Figure 19B shows a comparison of cell surface proteins between κAPC and MoDC, M1 MΦ and M2 MΦ, where Trem2, C3AR, IL-3R, LOX1, uPAR, CD40 and TLR2 were increased on κAPC compared to MoDC, M1 MΦ and M2 MΦ. Figure 19C shows pattern recognition receptors expressed by κAPC compared to M1 MΦ, where κAPC showed much higher levels of STING and TLR2. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0054] [Figure 20] FIG. 20 shows combined therapy of κAPC and tumor-localized RT against KPC pancreatic ductal adenocarcinoma.

[0055] [Figure 21-1] Figure 21A-B show therapeutic TT-κAPC vaccine against pancreatic cancer. Syngeneic KPC pancreatic ductal adenocarcinoma was established by subcutaneous engraftment. Figure 21A shows the experimental scheme and tumor volume changes in mice with and without TT-κAPC vaccination by intratumoral (it) injection. Figure 21B shows that TME analysis reveals a significantly increased population of CD45+ immune cells, especially CD8 T cells, in TT-κAPC vaccinated tumors associated with tumor regression. [Figure 21-2] Same as above.

[0056] [Figure 23-1] Figures 23A-D show NeoT cell targeting and clearance of multiple myeloma (MM) in vitro. Figure 23A shows the extent of NeoT cell proliferation after activation with κAPC or anti-CD3 / anti-CD28 antibody stimulation. Figure 23B provides light microscopy images of NeoT expansion after co-culture with MM antigen-loaded κAPC. Figure 23C shows MM-specific NeoT cells exhibiting potent cytolytic activity against autologous MM cells in whole bone marrow, avoiding non-myelogenous cells. NeoT activated by anti-CD3 / anti-CD28 antibody stimulation showed limited cytolytic efficacy against MM cells. Figure 23D provides still images from a time-lapse video of NeoT cells killing MM cells in in vitro co-culture. [Figure 23-2] Same as above. [Figure 23-3] Same as above.

[0057] [Figure 24-1]Figures 24A-C show κAPC activation of NeoT cells derived from ovarian tumor infiltrating lymphocytes (TILs). Figure 24A shows NeoT cell proliferation after activation with κAPC or anti-CD3 / anti-CD28 antibody stimulation, with NeoT responding continuously to κAPC activation but not to a second round of anti-CD3 / anti-CD28 antibody stimulation. Figure 24B compares CD4 and CD8 T cell counts in TILs before the addition of κAPC to the in vitro cultures, followed by 10 days of incubation with κAPC. After 10 days of proliferation, Neo-T showed significant proliferation in vitro. Figure 24C provides optical microscopy images of NeoT cells targeting dissociated ovarian cancer cells in vitro for cell lysis, which was recorded by time-lapse video. Graphical representation of the percentage of lysed ovarian cancer cells showed a significant increase in tumor-specific cytotoxicity of κAPC-activated Neo-T. [Figure 24-2] Same as above.

[0058] [Figure 25-1] FIG. 25 shows activation of autologous Neo T cells derived from TILs of various solid tumors (i.e., liver metastatic urothelial carcinoma, ovarian cancer, colon cancer, renal cancer, and lung cancer) by κ APC. [Figure 25-2] Same as above.

[0059] [Figure 26] FIG. 26 shows autologous NeoT cells engaging κ APCs followed by activation and clonally expanding in vitro.

[0060] [Figure 27-1]Figures 27A-C show the occurrence of Neo-T therapy for patients with metastatic uterine leiomyosarcoma. Figure 27A shows the differentiation of κAPC from autologous PBMC monocytes (cMo) with Karnelian X1 and Karnelian X2. κAPC showed strong antigen-presenting ability. Figure 27B shows the production of Neo-T in vitro. Figure 27C shows the Neo-T efficacy for killing cancer cells. A dose of approximately 2x107 Neo-T was administered intratumorally (it) and injected into the liver metastatic mass (upper panel), and this treatment induced tumor volume reduction when evaluated 4 weeks later (lower panel). [Figure 27-2] Same as above. [Figure 27-3] Same as above.

[0061] [Figure 28-1] Figures 28A-B show tumor progression as measured by change in tumor volume (Figure 28A) and overall survival (Figure 28B) in C57Bl / 6 mice engrafted with KPC pancreatic ductal adenocarcinoma that received NeoT-adapted cell therapy. [Figure 28-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description of the Invention This application provides novel compositions and agents that convert large numbers of monocytes, particularly those from cancer patients, into potent antigen-presenting cells (hereinafter referred to as "APCs"). These APCs can then be used to activate immune cells, providing highly effective therapeutic agents for the treatment of cancer.

[0063] The inventors have discovered that monocytes from cancer patients (referred to as "cMo" or "cMos") cannot or do not differentiate efficiently to become dendritic cell-like or macrophage-like APCs by conventional methods such as stimulation with M-CSF and / or GM-CSF. Such suppression can surprisingly be "unlocked" by cell culture supernatants of activated T cells containing high levels of IL-10. The inventors have surprisingly discovered that IL-10, which is generally considered to be immunosuppressive in nature, is essential to remove the immunosuppressive state of cMos. Without wishing to be bound by theory, IL-10 appears to provide differentiation factors (e.g., other cytokines) by activating the IL-10 receptor. It has also been found that IFNγ and TNFα present in the supernatant contribute to the survival of cancer monocytes, and IL-4, IFNγ and TNFα in the supernatant further promote the differentiation of monocytes into APCs. Based on these profound findings, the inventors have created a de novo composition comprising one or more of these important factors, and demonstrated that such a composition can achieve the same results as initially observed with the supernatant by combining an IL-10 receptor activator (such as IL-10) with one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator. APCs, when loaded with tumor-associated peptides (tumor cells / antigens), have been shown to perform antigen presentation and activate tumor antigen-specific CD4 and CD8 T cells, making them particularly effective in treating cancer.

[0064] Thus, the present application provides methods for generating APCs from monocytes (such as cMos), the use of APCs to activate immune cells, and the use of activated immune cells in the treatment of cancer. Compositions containing the key factors discussed herein are also provided.

[0065] Thus, the application provides, in one aspect, a method of stimulating a population of monocytes from an individual to produce a population of antigen presenting cells ("APCs") by separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby resulting in a population of APCs. The S / D / M factors need not perform the same function.

[0066] In another aspect, the application provides populations of APCs, such as APCs generated by some of the methods described above, and uses of the APCs for cancer treatment.

[0067] In another aspect, the present application provides a method for activating a population of immune cells (e.g., T cells), comprising co-culturing the population of immune cells with a population of APCs described herein, the APCs being pre-loaded with one or more neo-antigenic peptides. Also provided is a population of activated immune cells obtained by the method, and a method for treating cancer by administering the activated immune cells.

[0068] In another aspect, the application provides a composition comprising a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising: 1) an IL-10 receptor (IL-10R) activator; and 2) one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator.

[0069] I. Definition In general, the terms used in the claims and the specification are intended to be interpreted as having the plain meaning understood by a person skilled in the art. Certain terms are defined below to provide further clarity. In the event of a discrepancy between the plain meaning and the provided definition, the provided definition shall prevail.

[0070] The terms "individual," "subject," or "patient" are used interchangeably herein to describe a mammal, including a human. An individual includes, but is not limited to, a human, a bovine, an equine, a feline, a canine, a rodent, or a primate. In some embodiments, an individual is a human. In some embodiments, an individual is suffering from a disease, such as cancer. In some embodiments, an individual is in need of treatment.

[0071] "Monocyte," and "cell," as used herein, are understood to refer not only to the monocyte or cell from which it was obtained, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due, for example, to environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0072] "High level" or "higher level", "low level" or "lower level" when referring to the expression of a surface molecule on a population of cells (e.g., monocytes or APCs) refers to the average expression level of a particular surface molecule on a population of cells compared to the average level of the surface molecule on a reference cell population. Unless otherwise specified, the reference cell population refers to the corresponding cell population derived from a healthy donor. In some cases, a high level of a particular molecule is defined when the expression level of the molecule on the enumerated cell population is at least about 20% (e.g., 20%, 30%, 40%, 50% or more) higher than the expression level on the reference cell population. In some cases, a low level of a particular molecule is defined when the expression level of the molecule on the enumerated cell population is at least about 20% (e.g., 20%, 30%, 40%, 50% or more) lower than the expression level on the reference cell population.

[0073] "Reference" as used herein refers to any sample, standard, or level used for comparison purposes. Reference can be obtained from healthy and / or non-disease sample. In some examples, reference can be obtained from untreated sample. In some examples, reference can be obtained from non-disease or untreated sample of individual. In some examples, reference can be obtained from one or more healthy individuals who are not individual or patient.

[0074] As used herein, the term "antigen" is a substance that induces an immune response.

[0075] As used herein, the term "neo-antigen" refers to an antigen that has at least one change that makes it different from the corresponding wild-type parent antigen, for example, through a mutation in a tumor cell or a tumor cell-specific post-translational modification. A neo-antigen can include a polypeptide sequence. Mutations that result in a neo-antigen can include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, as well as any genomic or expression change that gives rise to a neo-ORF. Mutations can also include splice variants. Tumor cell-specific post-translational modifications can include aberrant phosphorylation. Tumor cell-specific post-translational modifications can also include proteasome-generated spliced ​​antigens. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21; 354(6310): 354-358.

[0076] As used herein, the term "tumor neo-antigen" or "cancer neo-antigen" is a neo-antigen that is present in tumor cells or tissues of a subject, but is not present in corresponding normal cells or tissues of the subject.

[0077] The term "peptide" refers to a polymer of 100 or less amino acids (including fragments of proteins), which may be linear or branched, may contain modified amino acids, and / or may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally occurring or modified by intervention, including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The term also includes, for example, polypeptides that contain one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. The peptides described herein may be naturally occurring, i.e., obtained or derived from a natural source (e.g., blood), or may be synthesized (e.g., chemically synthesized or synthesized by recombinant DNA technology).

[0078] As used herein, the term "epitope" is the particular portion of an antigen that is typically bound by an antibody or T-cell receptor.

[0079] As used herein, the term "immunogenic" is the ability to elicit an immune response, for example, via T cells, B cells, or both.

[0080] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity of binding between a particular antigen and a particular MHC allele.

[0081] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0082] As used herein, "activated T cells" refers to a population of monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., with clones encoding different TCRs) T cells that have a T cell receptor that recognizes at least one tumor antigen peptide. Activated T cells can include one or more subtypes of T cells, including, but not limited to, cytotoxic T cells (e.g., CD8 T cells), helper T cells (e.g., CD4 T cells), natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.

[0083] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the disease from worsening), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the appearance or recurrence of the disease, slowing or delaying the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving quality of life, and / or extending survival. Reduction of the pathological results of cancer is also encompassed by "treatment". The methods of the present invention contemplate one or more of these aspects of treatment.

[0084] As used herein, "delaying" the onset of cancer means to prolong, hinder, slow, retard, stabilize, and / or postpone the onset of the disease. This delay can be of various lengths of time, depending on the disease being treated and / or the medical history of the individual. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method of "delaying" the onset of cancer is one that reduces the probability of disease onset within a given time frame and / or reduces the extent of disease within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies with a statistically significant number of individuals. The onset of cancer can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriograms, or biopsies. Onset can also be initially undetectable and can refer to the progression of cancer, including emergence, recurrence, and onset.

[0085] As used herein, the term "co-administration" means that the first and second therapies in a combination therapy are administered at a time interval of about 15 minutes or less, such as about 10, 5, or 1 minute or less. When the first and second therapies are administered at the same time, the first and second therapies can be included in the same composition (e.g., a composition that includes both the first and second therapies) or in separate compositions (e.g., the first therapy in one composition and the second therapy are included in another composition).

[0086] As used herein, the term "sequential administration" means that the first and second therapies in the combination therapy are administered at a time interval of more than about 15 minutes, for example, more than about 20, 30, 40, 50, 60 minutes or more.Either the first or second therapy can be administered first.The first and second therapies are contained in separate compositions that can be contained in the same or different packages or kits.

[0087] As used herein, the term "concurrent administration" means that the administration of a first therapy and a second therapy in a combination therapy overlap with each other.

[0088] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to an individual without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharma-ceutically acceptable carrier or excipient preferably meets the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0089] It is understood that embodiments of the application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0090] Reference herein to "about" a value or parameter includes (and accounts for) the variation that is directed to the value or parameter itself. For example, a statement of "about X" includes the statement "X."

[0091] As used herein, a reference to a value or parameter "is not" generally means and describes a value or parameter "other than." For example, the method is not used to treat cancer type X means that the method is used to treat cancers other than type X.

[0092] As used herein, "about XY" is synonymous with "about X to about Y."

[0093] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0094] Terms not directly defined herein should be understood to have the meanings generally associated with them as understood within the technical field of the present invention. Certain terms are discussed herein to provide the practitioner with further guidance in describing the compositions, devices, methods, etc. of the present invention's embodiments and how to make or use them. It will be understood that the same thing may be said in two or more ways. Thus, alternative language and synonyms may be used in any one or more of the terms discussed herein. It is not important whether a term is detailed or discussed herein. Some synonyms or interchangeable methods, materials, etc. are provided. The listing of one or a few synonyms or equivalents does not exclude the use of other synonyms or equivalents unless expressly stated. The use of examples, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the present invention's embodiments herein.

[0095] II. Methods for Stimulating a Population of Monocytes to Generate APCs The present application provides various methods of stimulating a population of monocytes from an individual to produce a population of APCs.

[0096] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to promote monocyte survival, the method comprising contacting the population of monocytes with an IL-10 receptor (IL-10R) activator (e.g., IL-10, e.g., IL-12).

[0097] In some embodiments, methods are provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including one or more agents (e.g., two or more) selected from the group consisting of: 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), a TNFα receptor (TNFR) activator (e.g., TNFα), and an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs") by separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including two or more selected from the group consisting of 1) IL-10 and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), a TNFα receptor (TNFR) activator (e.g., TNFα), and an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), thereby obtaining a population of APCs. In some embodiments, the plurality of S / D / M factors are present in a single composition. In some embodiments, at least one of the plurality of S / D / M factors is provided separately from at least one other of the plurality of S / D / M factors. In some embodiments, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator (e.g., GM-CSF). In some embodiments, the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator (e.g., IL-6). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (e.g., 2-3 days).In some embodiments, the plurality of S / D / M factors are included in a composition derived from medium (e.g., supernatant) derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies. In some embodiments, the T cells are CD4 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the medium is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally for about 2 days.

[0098] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the multiple S / D / M factors include 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), and 3) a TNFα receptor (TNFR) activator (e.g., TNFα), and wherein the multiple S / D / M factors are present in a single composition, thereby resulting in a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10, 2) IL-4, 3) TNFα, and the plurality of S / D / M factors are present in a single composition, thereby obtaining a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days).In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0099] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors including 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), and 3) a TNFα receptor (TNFR) activator (e.g., TNFα), where at least the IL-4R activator is provided separately from the IL-10R activator or the TNFR activator, thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including 1) IL-10, 2) IL-4, 3) TNFα, where at least IL-4 is provided separately from IL-10 or TNFα, thereby obtaining a population of APCs. In some embodiments, an IL-4R activator is provided after an IL-10R activator is provided. In some embodiments, an IL-4R activator is provided after a TNFR activator is provided. In some embodiments, an IL-10R activator and a TNFR activator are provided simultaneously. In some embodiments, an IL-10R activator and a TNFR activator are provided sequentially. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα.In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0100] In some embodiments, methods are provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the multiple S / D / M factors include 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), and wherein the multiple S / D / M factors are present in a single composition, thereby resulting in a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10, 2) IL-4, 3) IFNγ, the plurality of S / D / M factors being present in a single composition, thereby obtaining a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml). In some embodiments, IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., from about 50 to 100 ng / ml).In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (e.g., 2-3 days). In some embodiments, the level of IL-10R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0101] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors including: 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), where at least the IL-4R activator is provided separately from the IL-10R activator or the IFNGR activator, thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including 1) IL-10, 2) IL-4, 3) IFNγ, where at least IL-4 is provided separately from IL-10 or IFNγ, thereby obtaining a population of APCs. In some embodiments, an IL-4R activator or IL-4 is provided after an IL-10R activator or IL-10 is provided. In some embodiments, an IL-4R activator or IL-4 is provided after an IFNGR activator or IFNγ is provided. In some embodiments, an IL-10R activator or IL-10 and an IFNGR activator or IFNγ are provided simultaneously. In some embodiments, the IL-10R activator or IL-10 and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml).In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0102] In some embodiments, methods are provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the multiple S / D / M factors include 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) a TNFα receptor (TNFR) activator (e.g., TNFα), and 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein the multiple S / D / M factors are present in a single composition, thereby resulting in a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10, 2) TNFα, and 3) IFNγ, the plurality of S / D / M factors being present in a single composition, thereby obtaining a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml).In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (e.g., 2-3 days). In some embodiments, the level of IL-10R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0103] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors including 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) a TNFα receptor (TNFR) activator (e.g., TNFα), and 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), where at least the IL-10R activator is provided separately from the TNFR activator or the IFNGR activator, thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors including 1) IL-10, 2) TNFα, and 3) IFNγ, where at least IL-10 is provided separately from TNFα or IFNγ, thereby obtaining a population of APCs. In some embodiments, the IL-10R activator or IL-10 is provided before the TNFR activator or TNFα is provided. In some embodiments, the IL-10R activator or IL-10 is provided before the IFNGR activator or IFNγ is provided. In some embodiments, the TNFR activator or TNFα and the IFNGR activator or IFNγ are provided simultaneously. In some embodiments, the TNFR activator or TNFα and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml).In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0104] In some embodiments, methods are provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the multiple S / D / M factors include 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) a TNFα receptor (TNFR) activator (e.g., TNFα), and 4) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), where the multiple S / D / M factors are present in a single composition, thereby resulting in a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10, and 2) IL-4, 3) TNFα, and 4) IFNγ, the plurality of S / D / M factors being present in a single composition, thereby obtaining a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., from about 50 to 100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα.In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0105] In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) a TNFα receptor (TNFR) activator (e.g., TNFα), and 4) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), where at least one of the plurality of S / D / M factors is provided separately from at least one other S / D / M factor of the plurality of S / D / M factors, thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10 and 2) IL-4, 3) TNFα, and 4) IFNγ, where at least one of the plurality of S / D / M factors is provided separately from at least one other S / D / M factor of the plurality of S / D / M factors, thereby obtaining a population of APCs. In some embodiments, the IL-10R activator or IL-10 is provided before the at least one other factor (e.g., the IL-4R activator or IL-4) is provided. In some embodiments, the IL-4R activator or IL-4 is provided after at least one other factor (e.g., an IL-10R activator or IL-10, an IFNGR activator or IFNγ, or a TNFGR activator or TNFα) is provided. In some embodiments, the IL-10R activator or IL-10, the TNFR activator or TNFα, and the IFNGR activator or TNFα are provided simultaneously.In some embodiments, the IL-10R activator or IL-10, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-4R activator or IL-4, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided simultaneously. In some embodiments, the IL-4R activator or IL-4, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (eg, 2-3 days).In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0106] In some embodiments, a method of stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs") comprises contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors being 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) a T A method is provided in which multiple S / D / M factors are present in a single composition, including NFα receptor (TNFR) activators (e.g., TNFα), 4) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), and 5) one or both of a GM-CSF receptor (GM-SCFR) activator (e.g., GM-CSF) and an IL-6 receptor (IL-6R) activator (e.g., IL-6), thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), either separately or simultaneously, the plurality of S / D / M factors comprising 1) IL-10 and 2) IL-4, 3) TNFα, 4) IFNγ, and 5) one or both of GM-CSF and IL-6, the plurality of S / D / M factors being present in a single composition, thereby obtaining a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., from about 50 to 100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα.In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally at about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally at about 100 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0107] In some embodiments, a method of stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs") comprises contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors being 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), 3) a TNFα receptor (TNFR) activator (e.g., IL-4R), 4) a TNFα receptor (TNFR) activator (e.g., IL-4R), 5) a TNFα receptor (TNFR) activator (e.g., IL-5), 6) a TNFα receptor (TNFR) activator (e.g., IL-5), 7) a TNFα receptor (TNFR) activator (e.g., IL-5), 8) a TNFα receptor (TNFR) activator (e.g., IL-5), 9) a TNFα receptor (TNFR) activator (e.g., IL-9), 10) a TNFα receptor (TNFR) activator (e.g., IL-10), 11) a TNFα receptor (TNFR) activator (e.g., IL-10), 12) a TNFα receptor (TNFR) activator (e.g., IL-12), 13) a TNFα receptor (TNFR) activator (e.g., IL-13), 14) a TNFα receptor (TNFR) activator (e.g., IL-14), 15) a TNFα receptor (TNFR) activator (e.g., IL-15), 16) a TNFα , TNFα), 4) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), and 5) one or both of a GM-CSF receptor (GM-SCFR) activator (e.g., GM-CSF) and an IL-6 receptor (IL-6R) activator (e.g., IL-6), wherein at least one of the plurality of S / D / M factors is provided separately from at least one other S / D / M factor of the plurality of S / D / M factors, thereby obtaining a population of APCs. In some embodiments, a method is provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising 1) IL-10, 2) IL-4, 3) TNFα, 4) IFNγ, and 5) one or both of GM-CSF and IL-6, where at least one of the plurality of S / D / M factors is provided separately from at least one other S / D / M factor of the plurality of S / D / M factors, thereby obtaining a population of APCs. In some embodiments, the IL-10R activator or IL-10, and / or the GM-CSFR activator or GM-CSF is provided before at least one other factor (e.g., an IL-4R activator or IL-4, e.g., an IL-6R activator or IL-6) is provided.In some embodiments, the IL-4R activator or IL-4, and / or the IL-6R activator or IL-6 are provided after at least one other factor (e.g., an IL-10R activator or IL-10, e.g., a GM-CSFR activator or GM-CSF) is provided. In some embodiments, the IL-10R activator or IL-10, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided simultaneously. In some embodiments, the IL-10R activator or IL-10, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-4R activator or IL-4, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided simultaneously. In some embodiments, the IL-4R activator or IL-4, the TNFR activator or TNFα, and the IFNGR activator or IFNγ are provided sequentially. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml). In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4.In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally at about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (e.g., 2-3 days). In some embodiments, the level of IL-10R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0108] In some embodiments, methods are provided for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the multiple S / D / M factors include 1) IL-10 (e.g., human IL-10 or human recombinant IL-10) and 2) IL-4 (e.g., human IL-4 or human recombinant IL-4), 3) TNFα (e.g., human TNFα or human recombinant TNFα), 4) IFNγ (e.g., human IFNγ or human recombinant IFNγ), and 5) GM-CSF (e.g., human GM-CSF or human recombinant GM-CSF), and 6) IL-6 (e.g., human IL-6 or human recombinant IL-6), where the multiple S / D / M factors are present in a single composition, thereby resulting in a population of APCs. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml).In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally at about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally at about 100 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0109] In some embodiments, a method for stimulating a population of monocytes from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells ("APCs") includes contacting the population of monocytes separately or simultaneously with multiple survival, differentiation and / or maturation factors ("S / D / M factors"), the multiple S / D / M factors being 1) IL-10 (e.g., human IL-10 or human recombinant IL-10) and 2) IL-4 (e.g., human IL-4 or human recombinant IL-4), 3) TNFα (e.g., human T and 5) GM-CSF (e.g., human GM-CSF or human recombinant GM-CSF), and 6) IL-6 (e.g., human IL-6 or human recombinant IL-6), where at least one of the plurality of S / D / M factors is provided separately from at least one other of the plurality of S / D / M factors, thereby obtaining a population of APCs. In some embodiments, IL-10 and / or GM-CSF are provided before at least one other factor (e.g., IL-4 or IL-6) is provided. In some embodiments, IL-4 and / or IL-6 are provided after at least one other factor (e.g., IL-10 or GM-CSF) is provided. In some embodiments, IL-10, TNFα, and IFNγ are provided simultaneously. In some embodiments, IL-10, TNFα, and IFNγ are provided sequentially. In some embodiments, IL-4, TNFα and IFNγ are provided simultaneously. In some embodiments, IL-4, TNFα and IFNγ are provided sequentially. In some embodiments, IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, IFNγ is human IFNγ or human recombinant IFNγ.In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally at about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally at about 100 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1 to 3 days (e.g., 2 to 3 days). In some embodiments, the level of IL-10R on monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual).In some embodiments, the level of IL-4R on monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0110] In some embodiments, a method is provided for stimulating a population of monocytes from an individual with cancer to produce a population of antigen presenting cells ("APCs"), comprising contacting the population of monocytes with medium derived from a culture (e.g., supernatant) of T cells after treatment with anti-CD3 and anti-CD28 antibodies, wherein the medium comprises an IL-10R activator (e.g., IL-10). In some embodiments, the medium further comprises an IL-4R activator (e.g., IL-4), an IFNGR activator (e.g., IFNγ), a TNFR activator (e.g., TNFα). In some embodiments, the medium further comprises a GM-CSFR activator (e.g., GM-CSF) and / or an IL-6R activator (e.g., IL-6). In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the medium is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally for about 2 days. In some embodiments, the monocytes are cultured for about 2-3 days in the presence of medium derived from a culture of T cells. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml).In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally at about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml). In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally at about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml). In some embodiments, the monocytes are cultured in the presence of at least one of the S / D / M factors for about 1-3 days (e.g., 2-3 days). In some embodiments, the level of IL-10R on the monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-4R on monocytes prior to contact with the S / D / M factors is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0111] In some embodiments according to any of the embodiments described above, the method further comprises contacting the population of monocytes with a plurality of refinement factors selected from the group consisting of a type I interferon, IFNγ, TNFα, a TLR ligand, CD40L or CD40 ligating antibody, an anti-PD-L1 antibody, and TPI-1, optionally wherein the type I interferon comprises IFNα and / or IFNβ, and optionally wherein the TLR ligand is Poly IC, CpG, or LPS. In some embodiments, the plurality of refinement factors is provided after contacting the plurality of monocytes with a plurality of S / D / M factors or medium derived from a culture of T cells, thereby producing a population of APCs, and wherein the population of APCs is cultured in the presence of the plurality of refinement factors for about 1-5 days, and optionally wherein the population of APCs is cultured for about 1 day. In some embodiments, multiple refining factors are provided when a) at least about 50% of the monocytes are viable, b) at least about 30% of the population of APCs exhibit dendritic cell morphology, and / or c) the population of APCs expresses i) high levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86 and / or CD40, and / or ii) low levels of SIRPα.

[0112] In some embodiments, the method further comprises contacting the population of monocytes with a plurality of refining factors, including IFNα, IFNγ and TNFα.

[0113] In some embodiments, refinement factors include IFNα, IFNγ, TNFα, Poly IC, CpG.

[0114] In some embodiments, refinement factors include IFNα, IFNγ, TNFα, Poly IC, CpG, CD40L and anti-PD-L1 antibody.

[0115] In some embodiments, refinement factors include IFNα, IFNγ, TNFα, Poly IC, CpG, CD40L, TPI-1 and anti-PD-L1 antibody.

[0116] Survival, Differentiation and / or Maturation Factors ("S / D / M Factors") In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein comprise one or more agents selected from the group consisting of: 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) an IL-4 receptor (IL-4R) activator (e.g., IL-4), a TNFα receptor (TNFR) activator (e.g., TNFα), and an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ).

[0117] In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein comprises 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) IFNγ. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein comprises 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) TNFα. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein comprises 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) IL-6. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein comprises 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) IL-4. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein includes 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) GM-CSF. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein includes 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) IL-12. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein includes 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) Poly IC. In some embodiments, the plurality of survival, differentiation and / or maturation factors ("S / D / M factors") described herein includes 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10) and 2) CpG.

[0118] In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein include 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), 2) a TNFα receptor (TNFR) activator (e.g., TNFα), and 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ).

[0119] In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein include 1) IL-10 receptor (IL-10R) activators (e.g., IL-10), 2) TNFα receptor (TNFR) activators (e.g., TNFα), 3) interferon gamma (IFNγ) receptor (IFNGR) activators (e.g., IFNγ), 4) IL-6 receptor (IL-6R) activators (e.g., IL-6), and 5) GM-CSF receptor (GM-CSF) activators (e.g., GM-CSF).

[0120] In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein include 1) IL-22, 2) TNFα receptor (TNFR) activators (e.g., TNFα), 3) interferon gamma (IFNγ) receptor (IFNGR) activators (e.g., IFNγ), 4) IL-6 receptor (IL-6R) activators (e.g., IL-6), and 5) GM-CSF receptor (GM-CSF) activators (e.g., GM-CSF).

[0121] In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein include 1) TNFα receptor (TNFR) activators (e.g., TNFα), 2) interferon gamma (IFNγ) receptor (IFNGR) activators (e.g., IFNγ), 3) IL-6 receptor (IL-6R) activators (e.g., IL-6), and 4) GM-CSF receptor (GM-CSF) activators (e.g., GM-CSF).

[0122] In some embodiments, the multiple survival, differentiation and / or maturation factors ("S / D / M factors") described herein include 1) IL-10 receptor (IL-10R) activators (e.g., IL-10), 2) TNFα receptor (TNFR) activators (e.g., TNFα), 3) interferon gamma (IFNγ) receptor (IFNGR) activators (e.g., IFNγ), 4) IL-6 receptor (IL-6R) activators (e.g., IL-6), 5) IL-4 receptor (IL-4R) activators (e.g., IL-4), and 6) GM-CSF receptor (GM-CSF) activators (e.g., GM-CSF).

[0123] IL-10 receptor (IL-10R) activators and IL-10 As used herein, "IL-10 receptor (IL-10R) activator" refers to a molecule that activates the IL-10 receptor-mediated signaling pathway. IL-10R includes both IL-10R1 and IL-10R2.

[0124] Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine. In humans, interleukin 10 is encoded by the IL10 gene. IL-10 signals through a receptor complex consisting of two IL-10 receptor-1 and two IL-10 receptor-2 proteins. As a result, the functional receptor consists of four IL-10 receptor molecules. IL-10 binding induces STAT3 signaling via phosphorylation of the cytoplasmic tails of IL-10 receptor 1 and IL-10 receptor 2 by JAK1 and Tyk2, respectively. See, e.g., Saraiva, M., O'Garra, A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 10, 170-181 (2010).

[0125] In humans, IL-10 is encoded by the IL10 gene, which is located on chromosome 1 and contains five exons, and is produced primarily by monocytes and, to a lesser extent, lymphocytes, namely type II T helper cells (TH2), mast cells, CD4+CD25+Foxp3+ regulatory T cells, and certain subsets of activated T and B cells. IL-10 can be produced by monocytes upon PD-1 triggering in these cells.

[0126] IL-10 is a cytokine that has multiple pleiotropic effects in immune regulation and inflammation. It downregulates the expression of Th1 cytokines, MHC class II antigens, and costimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity and participates in the regulation of the JAK-STAT signaling pathway.

[0127] IL-10 was initially reported to suppress cytokine secretion, antigen presentation, and CD4+ T cell activation. Further investigations showed that IL-10 primarily inhibited lipopolysaccharide (LPS)- and bacterial product-mediated induction of proinflammatory cytokines TNFα, IL-1β, IL-12, and IFNγ secretion from Toll-like receptor (TLR)-induced myeloid cells.

[0128] IL-10 as referred to herein includes any construct having a component of IL-10 (e.g., naturally occurring IL-10, e.g., recombinant IL-10). These include, but are not limited to, natural IL-10 (e.g., various isoforms of human IL-10), synthetic or recombinant IL-10, and fusion proteins having an IL-10 component.

[0129] In some embodiments, the IL-10R activator further comprises a stability- or half-life-enhancing moiety, including, for example, an Fc moiety or a PEG moiety.

[0130] In some embodiments, the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). In some embodiments, the IL-10R activator is IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 (e.g., human IL-10) is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml (e.g., about 20 ng / ml). In some embodiments, IL-10 (e.g., human IL-10) is present in the medium at a concentration of about 2 ng / ml to about 200 ng / ml (e.g., about 10 ng / ml to about 200 ng / ml, e.g., about 10 ng / ml to about 100 ng / ml, e.g., about 20 ng / ml to about 100 ng / ml).

[0131] STAT3 activators and STAT3 In some embodiments, the IL-6R activator is a STAT3 activator. As used herein, "STAT3 activator" refers to a molecule that activates STAT3 signaling, such as STAT3 nuclear localization and transcription factor activity.

[0132] STAT3 is a transcription factor that resides in the cytoplasm in its inactive, non-phosphorylated form and translocates to the nucleus upon its activation via phosphorylation, e.g., Tyr705 phosphorylation and subsequent dimerization. Upon entry into the nucleus, the activated STAT3 dimer binds to IFN-γ activating sequences (GAS) in target promoters, thereby activating the transcription of target genes. Multiple tyrosine kinases have been described as intracellular activators of STAT3 activity (e.g., JAK1, JAK2, EGFR, Src and ERK). Further mechanisms of activation include i) STAT3 phosphorylation at Ser727 by protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and CDK5; and ii) STAT3 acetylation at Lys685 by histone acetyltransferases, which can increase the stability of STAT3 dimers. See, e.g., Rebe et al., JAKSTAT 2013;2(1):e23010.

[0133] STAT3 is expressed in most cell types under certain conditions and has been described to be generally involved in biological processes such as cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, inflammation and immunity. In immune cells, STAT3 has been described in contradictory terms. For example, STAT3 has been described to promote the differentiation of macrophages into the M2 phenotype and the absence of functional dendritic cells (see, e.g., Rebe et al., 2013). STAT3 has also been described to promote gp130-mediated maintenance of the pluripotent state of proliferating embryonic stem cells and promote gp130-induced macrophage differentiation of M1 cells. Both c-myc and pim have been identified as target genes of STAT3 and together can compensate for STAT3 in cell survival and cell cycle transition (see, e.g., Hirano et al. Oncogene 2000;19:2548-2556).

[0134] STAT3 activation is rapid and transient under normal biological conditions and is mediated by many extracellular stimuli, including cytokines (IL-6, IL-10, IFN, TNFα, LIF, OSM, etc.) and growth factors (e.g., EGF, G-CSF, GM-CSF, VEGF, HGF, GH, and Her2 / Neu). Active oncogenic proteins such as Src (e.g., v-Src) and Ras, as well as chemical carcinogens and other molecules, can also activate STAT3. In fact, many regulatory genes induced by STAT3 activity also activate the same STAT3 pathway, thereby maintaining a stable feed-forward loop. In some embodiments, the STAT3 activator comprises a cytokine selected from the group consisting of IL-6, IL-10, IL-11, IL-12, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, IL-27, IFNα, IFNβ, IFNγ, TNFα, leukemia inhibitory factor (LIF), oncostatin M (OSM), biologically active derivatives thereof, and any combination thereof. In some embodiments, the STAT3 activator comprises a growth factor selected from the group consisting of EGF, FGF, IGF, G-CSF, GM-CSF, VEGF, HGF, GF, Her2 / Neu, biologically active derivatives thereof, and any combination thereof. In some embodiments, the STAT3 activator comprises a JAK activator, such as an enzyme that phosphorylates JAK (e.g., JAK2). In some embodiments, the STAT3 activator comprises a hormone (e.g., leptin). In some embodiments, the STAT3 activator comprises a chaperone protein (e.g., HSP90, HSP70, HSP27, HSP110, HOP).

[0135] STAT3 activity can be positively regulated by the signaling pathway of IL-10 and IL-10 family members, including IL-19, IL-20, IL-22, IL-24 and IL-26. In addition, IL-12 and related family members (e.g., IL-23) can activate STAT3 activity, at least in part, by promoting IL-10 / IL-10R production and autocrine signaling in κAPC cells. IL-6 has also been described as an activator of the STAT3 pathway. In some embodiments, the STAT3 activator is selected from the group consisting of IL-6, IL-10, IL-12, IL-19, IL-20, IL-22, IL-23, IL-24, IL-26, biologically active derivatives thereof, and any combination thereof. In some embodiments, the STAT3 activator is an IL-10R activator, such as any of those described herein or known in the art.

[0136] In some embodiments, the IL-10R activator is an activator of IL-10R downstream STAT3. STAT3 activators can include, but are not limited to, any of the following: small molecules, nucleic acids (e.g., siRNA, shRNA, antisense RNA, microRNA), nucleobase inhibitors (e.g., circular RNA inhibitors), nucleic acid editing systems (e.g., CRISPR, ZFN or TALENS systems), decoy oligonucleotides, peptide agents, protein agents (e.g., antibody agents targeting IL-10R; e.g., protein agents that target STAT3 phosphorylation and / or prevent STAT3 dephosphorylation), protein stabilizers (e.g., STAT3 stabilizers such as chaperone proteins, e.g., HSP90, HSP70, HSP27, HSP110 and / or HOP), protein degradation or destabilization agents (e.g., phosphatase degradation or destabilization agents such as phosphatase-targeted PROTACs, LYTACs, molecular adhesives, AbTACs, CMATACs), proteins modified with unnatural amino acids, viral agents (e.g., Kaposi's Sarcoma Herpes Virus (KSHV)), derivatives thereof, and any combination thereof.

[0137] In some embodiments, the STAT3 activator comprises a cancer cell STAT3 activator. These cancer cell STAT3 activators can include any of PVT1, NEAT1, FEZF1-AS1, UICC, MALAT1, XIST, miR-30d, CD109, CD146, CD24, CDK7, SOX, Smad6, Smad7, TRIM24, TRIM27, TRIM59, ADAM12, USP22, BMX AKR1C1, PRMT1, PBX1, HSP110, RanBP6, RAC1-GTP, PA28γ, E6, and FABP5.

[0138] In some embodiments, the STAT3 activator comprises a small molecule selected from the group consisting of colivelin, colivelin TFA, garcinone D, butizamide, efrepedoquin alpha, brusonine E, derivatives thereof, and any combination thereof. In some embodiments, the STAT3 activator comprises colivelin, colivelin TFA, and / or garcinone D.

[0139] In some embodiments, STAT3 activators include inhibitors or antagonists of molecules or compounds that inactivate STAT3 (e.g., reduce phosphorylated STAT3 levels) or reduce total STAT3 levels (e.g., via proteasomal degradation and / or transcriptional repression) in cells of interest, such as myeloid cells, such as κAPC cells. For example, molecules or compounds that can inactivate STAT3 include, but are not limited to, β-elemene, selective serotonin reuptake inhibitors (SSRIs, e.g., fluoxetine), minicoside, luteolin (3,4,5,7-tetrahydroxyflavone), SHP-1, SHP-2, PTP1B, PTPRM, eEF2 kinase, PKM2, curcumin, cucurbitacin, honokiol, gugulasterone, resveratrol, berbamine, flavopiridol, JAK inhibitors / inactivating JAK (e.g., JAK2), low molecular weight-DSP2, PIAS3, etc. In some embodiments, molecules or compounds that can reduce total STAT3 levels (e.g., via proteasomal degradation and / or transcriptional repression) include, but are not limited to, PDLIM2, COP1, calcineurin, SOCS proteins, rubulavirus (e.g., mumps virus, e.g., mumps virus V protein, e.g., V-dependent degradation complex VDC or V / DDB1 / cullin degradation complex), TSM-1, KYM-003, KTX-201, SD-36, AUY922, 17-DMAG, etc. In some embodiments, inhibitors of molecules or compounds that inactivate STAT3 or reduce total STAT3 levels in a cell of interest competitively bind to STAT3, preventing inactivation (e.g., DDIAS) or proteolysis (e.g., chaperones such as HSP90).

[0140] See, e.g., Kim et al. Oncol Lett. 2022;23(3):94; Zheng et al. Exp Mol Med. 2018;50(9):1-14; Liao et al. Anticancer Res. 2022;42(8):3807-3814; Xiao et al. Cell Commun Signal. 2020;18(1):25; Jego et al. Cancers (Basel) 2020;12(1):21; Liu et al. Cell Death Dis. 2014;5(6):e1293; and Yang et al. Cytokine Growth Factor Rev. 2019;46:10-22.

[0141] Exemplary amounts of STAT3 activators can be found, for example, in Table 1.

[0142] IL-4 receptor (IL-4R) activator and IL-4 As used herein, "IL-4 receptor (IL-4R) activator" refers to a molecule that activates the IL-4 receptor-mediated signaling pathway.

[0143] Interleukin 4 (IL-4) is a cytokine that induces the differentiation of naive helper T cells (Th0 cells) into Th2 cells. When activated by IL-4, Th2 cells then produce additional IL-4 in a positive feedback loop. IL-4 is produced primarily by mast cells, Th2 cells, eosinophils and basophils. It is closely related and has similar functions to IL-13. Interleukin 4 has many biological roles, including stimulation of activated B and T cell proliferation, and differentiation of B cells into plasma cells. It is a key regulator in humoral and adaptive immunity. IL-4 induces the switch of B cell classes to IgE and upregulates MHC class II production. IL-4 reduces the production of IL-12 in Th1 cells, macrophages, IFNγ and dendritic cells. IL-4 signaling determines the level of CD20 on the surface of normal and malignant B lymphocytes through activation of the transcription factor STAT6. Overproduction of IL-4 is associated with allergies.

[0144] IL-4 as referred to herein includes any construct having a component of IL-4 (e.g., naturally occurring IL-4, e.g., recombinant IL-4). These include, but are not limited to, natural IL-4 (e.g., various isoforms of human IL-4), synthetic or recombinant IL-4, and fusion proteins having an IL-4 component.

[0145] In some embodiments, the IL-4R activator further comprises a stability- or half-life-enhancing moiety, including, for example, an Fc moiety or a PEG moiety.

[0146] In some embodiments, the plurality of S / D / M factors comprises an IL-4R activator, optionally the IL-4R activator is selected from the group consisting of IL-4, IL-13, an IL-4R agonist antibody, and a small molecule activator of IL-4R. In some embodiments, the IL-4R activator is IL-4. In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 (e.g., human IL-4) is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml (e.g., at least about 30 pg / ml, 50 pg / ml, 75 pg / ml, 100 pg / ml, 125 pg / ml, or 150 pg / ml). In some embodiments, IL-4 (e.g., human IL-4) is present in the medium at a concentration of about 15 pg / ml to about 1.5 ng / ml (e.g., about 30 pg / ml to about 1 ng / ml, e.g., about 100 pg / ml to about 1 ng / ml, e.g., about 100 pg / ml to about 1 ng / ml).

[0147] In some embodiments, the IL-4R activator is IL-13 (such as human IL-13 or human recombinant IL-13). In some embodiments, IL-13 is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 60 pg / ml, and further optionally from about 60 pg / ml to about 2 ng / ml (e.g., from about 100 pg / ml to about 2 ng / ml).

[0148] TNFα receptor (TNFR) activators and TNFα As used herein, a "TNFα receptor (TNFR) activator" refers to a molecule that activates a TNFR-mediated signaling pathway. As used herein, TNFR refers to either TNFR1 or TNFR2.

[0149] Tumor necrosis factor α (TNF, cachexin or cachectin; often called tumor necrosis factor alpha or TNF-α) is an adipokine and cytokine. TNFα is a member of the TNFα superfamily, which consists of various transmembrane proteins with homologous TNFα domains.

[0150] TNFα can bind to two receptors, TNFR1 (TNFα receptor type 1; CD120a; p55 / 60) and TNFR2 (TNFα receptor type 2; CD120b; p75 / 80). TNFR1 is 55 kDa and TNFR2 is 75 kDa.

[38] TNFR1 is expressed in most tissues and can be fully activated by both membrane-bound and soluble trimeric forms of TNF, whereas TNFR2 is typically found on cells of the immune system and responds to membrane-bound TNFα homotrimers.

[0151] TNFα was thought to be produced primarily by macrophages,

[50] but is also produced by a wide variety of cell types, including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts, and neurons.

[51] [Unreliable medical source?] Large amounts of TNFα are released in response to lipopolysaccharide, other bacterial products, and interleukin-1 (IL-1). In the skin, mast cells appear to be the major source of preformed TNFα that can be released upon inflammatory stimuli (e.g., LPS).

[0152] TNFα as referred to herein includes any construct having a component of TNFα (e.g., naturally occurring TNFα, e.g., recombinant TNFα). These include, but are not limited to, natural TNFα (e.g., various isoforms of human TNFα), synthetic or recombinant TNFα, and fusion proteins having a TNFα component.

[0153] In some embodiments, the TNFR activator further comprises a stability or half-life enhancing moiety, including, for example, an Fc moiety or a PEG moiety.

[0154] In some embodiments, the plurality of S / D / M factors includes a TNFR activator, and optionally the TNFR activator is selected from the group consisting of TNFα, TNFR agonist antibodies, and small molecule activators of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα (e.g., human TNFα) is present in the medium at a concentration of at least about 0.2 ng / ml, optionally at least about 0.5 ng / ml (e.g., at least about 1 ng / ml, about 2 ng / ml, or about 3 ng / ml). In some embodiments, the TNFα (e.g., human TNFα) is present in the medium at a concentration of about 0.2 ng / ml to about 30 ng / ml (e.g., about 0.5 ng / ml to about 10 ng / ml, e.g., about 1 ng / ml to about 5 ng / ml, e.g., about 2 ng / ml to about 4 ng / ml).

[0155] IFN-γ receptor (IFNGR) activators and IFN-γ As used herein, "IFNγ receptor (INFGR) activator" refers to a molecule that activates the INFGR-mediated signaling pathway.

[0156] Interferon gamma (IFN-γ) is a dimerizing soluble cytokine that is the only member of the type II class of interferons. IFN-γ or type II interferons are cytokines important in innate and adaptive immunity against viral, some bacterial and protozoan infections. IFN-γ is an important activator of macrophages and an inducer of major histocompatibility complex class II molecule expression. Aberrant IFN-γ expression is associated with several autoinflammatory and autoimmune diseases. The importance of IFN-γ in the immune system is due in part to its ability to directly inhibit viral replication and, most importantly, to its immunostimulatory and immunomodulatory effects. IFN-γ is primarily produced by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops as part of the adaptive immune response. IFN-γ is also produced by non-cytotoxic innate lymphoid cells (ILCs), a family of immune cells first discovered in the early 2010s.

[0157] IFN-γ as referred to herein includes any construct having a component of IFN-γ (e.g., naturally occurring IFN-γ, e.g., recombinant IFN-γ). These include, but are not limited to, natural IFN-γ (e.g., various isoforms of human IFN-γ), synthetic or recombinant IFN-γ, and fusion proteins having an IFN-γ component.

[0158] In some embodiments, the IFNGR activator further comprises a moiety that enhances stability or half-life (eg, including an Fc moiety or a PEG moiety).

[0159] In some embodiments, the plurality of S / D / M factors comprises an IFNGR activator, and optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR. In some embodiments, the IFNGR activator is IFNγ. In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ (e.g., human IFNγ) is present in the medium at a concentration of at least about 1 ng / ml, optionally at least about 5 ng / ml (e.g., at least about 10 ng / ml, about 20 ng / ml, or about 50 ng / ml). In some embodiments, IFNγ (e.g., human IFNγ) is present in the medium at a concentration of about 1 ng / ml to about 500 ng / ml (e.g., about 5 ng / ml to about 200 ng / ml, e.g., about 10 ng / ml to about 100 ng / ml, e.g., about 40 ng / ml to about 60 ng / ml, e.g., about 50 ng / ml).

[0160] In some embodiments, the multiple S / D / M factors include two or more agents selected from the group consisting of an IL-4R activator, a TNFR activator, and an IFNGR activator as described herein.

[0161] In some embodiments, the multiple S / D / M factors include IL-10, IL-4, TNFα, and IFNγ.

[0162] GM-CSF receptor (GM-CSFR) activators and GM-CSF As used herein, a "GM-CSF receptor (GM-CSFR) activator" refers to a molecule that activates the GM-CSFR-mediated signaling pathway.

[0163] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts, where it functions as a cytokine. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, basophils) and monocytes. Monocytes leave the circulation and migrate to tissues, where they subsequently mature into macrophages and dendritic cells. This is part of the immune / inflammatory cascade, whereby activation of a small number of macrophages rapidly leads to an increase in their numbers, a process that is important for fighting infections. GM-CSF also has several effects on mature cells of the immune system. These include, for example, enhancing neutrophil migration and causing changes in receptors expressed on the cell surface.

[0164] GM-CSF as referred to herein includes any construct having a component of GM-CSF (e.g., naturally occurring GM-CSF, such as recombinant GM-CSF). These include, but are not limited to, native GM-CSF (e.g., various isoforms of human GM-CSF), synthetic or recombinant GM-CSF, and fusion proteins having a GM-CSF component.

[0165] In some embodiments, the GM-CSFR activator further comprises a stability or half-life enhancing moiety, including, for example, an Fc moiety or a PEG moiety.

[0166] In some embodiments, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator. In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF (e.g., human GM-CSF) is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml (e.g., at least about 100 pg / ml, about 150 pg / ml, about 200 pg / ml, or about 300 pg / ml). In some embodiments, GM-CSF (e.g., human GM-CSF) is present in the medium at a concentration of about 30 pg / ml to about 3 ng / ml (e.g., about 50 pg / ml to about 1 ng / ml, e.g., about 100 pg / ml to about 500 pg / ml, e.g., about 200 pg / ml to about 400 pg / ml, e.g., about 300 pg / ml).

[0167] IL-6 receptor (IL-6R) activator and IL-6 As described herein, "IL-6 receptor (IL-6R) activator" refers to a molecule that activates the IL-6 receptor-mediated signaling pathway.

[0168] Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. In the immune system, IL-6 is secreted by macrophages in response to certain microbial molecules called pathogen-associated molecular patterns (PAMPs). These PAMPs bind to an important group of detector molecules of the innate immune system called pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and in intracellular compartments and induce intracellular signaling cascades that lead to inflammatory cytokine production. IL-6 is a key mediator of fever and acute phase responses. IL-6 is responsible for stimulating acute phase protein synthesis as well as the production of neutrophils in the bone marrow. It supports B cell growth and is antagonistic to regulatory T cells.

[0169] IL-6 as referred to herein includes any construct having a component of IL-6 (e.g., naturally occurring IL-6, e.g., recombinant IL-6). These include, but are not limited to, natural IL-6 (e.g., various isoforms of human IL-6), synthetic or recombinant IL-6, and fusion proteins having an IL-6 component.

[0170] In some embodiments, the IL-6R activator further comprises a stability- or half-life-enhancing moiety, including, for example, an Fc moiety or a PEG moiety.

[0171] In some embodiments, the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R. In some embodiments, the IL-6R activator is IL-6. In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 (e.g., human IL-6) is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml (e.g., at least about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, or about 25 pg / ml). In some embodiments, IL-6 (e.g., human IL-6) is present in the medium at a concentration of about 1 pg / ml to about 300 pg / ml (e.g., about 5 pg / ml to about 100 pg / ml, e.g., about 10 pg / ml to about 50 pg / ml, e.g., about 20 pg / ml to about 40 pg / ml, e.g., about 30 pg / ml).

[0172] In some embodiments, the multiple S / D / M factors include IL-10, IL-4, TNFα, IFNγ, GM-CSF and IL-6.

[0173] In some embodiments, multiple S / D / M factors described herein are present in a single composition.

[0174] In some embodiments, the multiple S / D / M factors described herein further comprise one or more cytokines selected from the group consisting of IL-2, IL-17 (e.g., IL-17A), and / or M-CSF.

[0175] In some embodiments, at least one of the plurality of S / D / M factors (e.g., IL-10) is provided separately from other S / D / M factors of the plurality of S / D / M factors.

[0176] Refining Factor In some embodiments, the method further comprises contacting the population of monocytes with a plurality of refining factors after contacting the plurality of monocytes with the plurality of S / D / M factors or medium from a culture of T cells, the refining factors being selected from the group consisting of type I interferons (such as IFNα and / or IFNβ), IFNγ, TNFα, TLR ligands (such as polyIC, CpG, or LPS), CD40L or CD40 ligation antibodies, anti-PD-L1 antibodies, and TPI-1.

[0177] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNα, b) IFNγ, and c) TNFα.

[0178] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) TNFα, d) Poly IC, and e) CpG.

[0179] In some embodiments, there is provided a method of refining a population of APCs comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) TNFα, d) Poly IC, e) CD40L, and f) an anti-PD-L1 antibody.

[0180] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) TNFα, d) Poly IC, e) CD40L, f) an anti-PD-L1 antibody, g) a SHP-1 inhibitor (e.g., TPI-1).

[0181] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) R848, d) Poly IC, or e) a SHP-1 inhibitor (e.g., TPI-1).

[0182] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNγ, b) R848, c) Poly IC, and d) a SHP-1 inhibitor (e.g., TPI-1).

[0183] In some embodiments, a method of refining a population of APCs is provided, the method comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) Poly IC, d) CpG, e) CD40L, f) an anti-PD-L1 antibody, g) a SHP-1 inhibitor (e.g., TPI-1), and h) TNFα.

[0184] In some embodiments, the refinement factors are provided immediately after contacting the monocytes with the S / D / M factors or medium from a culture of T cells, hi some embodiments, the refinement factors are provided within about one day after contacting the monocytes with the S / D / M factors or medium from a culture of T cells.

[0185] In some embodiments, the monocytes are cultured in the presence of the refining factors for about 1 to 5 days (eg, about 1, 2, 3, 4, or 5 days).

[0186] In some embodiments, the refinement factors are provided when at least about 50% (e.g., about 50%, 60%, 70%, 80%, or 99%) of the monocytes survive after the monocytes are contacted with the S / D / M factors or medium derived from a culture of T cells.

[0187] In some embodiments, multiple refining factors are provided when at least about 10%, 20%, 30%, 40% or 50% of the monocytes exhibit dendritic cell morphology.

[0188] In some embodiments, multiple refinement factors are provided when monocytes express high levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86, and / or CD40. In some embodiments, multiple refinement factors are provided when monocytes express higher (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86, and / or CD40 than monocytes obtained from the same individual and cultured with GM-CSF and M-CSF (e.g., at concentrations routinely used in the art for such methods).

[0189] In some embodiments, multiple refinement factors described herein may be used independently of the above methods for priming APCs (eg, APCs obtained from humans).

[0190] In some embodiments, the refinement factors include IFNα, IFNγ, and TNFα.

[0191] In some embodiments, the refinement factors include IFNα, IFNγ, TNFα, Poly IC and CpG.

[0192] In some embodiments, refinement factors include IFNα, IFNγ, TNFα, Poly IC, CpG, CD40L and anti-PD-L1 antibody.

[0193] In some embodiments, the refinement factors include IFNα, IFNγ, TNFα, Poly IC, CpG, CD40L, anti-PD-L1 antibody, and TPI-1.

[0194] In some embodiments, the concentration of IFNα in the refinement factor is about 1 ng / ml to about 50 ng / ml (eg, about 5 ng / ml to about 20 ng / ml, for example, about 10 ng / ml).

[0195] In some embodiments, the concentration of IFNγ in the refinement factor is about 5 ng / ml to about 500 ng / ml (e.g., about 10 ng / ml to about 250 ng / ml, e.g., about 20 ng / ml to about 100 ng / ml, e.g., about 50 ng / ml).

[0196] In some embodiments, the concentration of TNFα in the refinement factor is about 1 ng / ml to about 50 ng / ml (eg, about 5 ng / ml to about 20 ng / ml, for example, about 10 ng / ml).

[0197] In some embodiments, the concentration of Poly IC in the refinement agent is about 0.1 μg / ml to about 10 μg / ml (e.g., about 0.2 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml to about 2.5 μg / ml, e.g., about 1 μg / ml).

[0198] In some embodiments, the concentration of CpG in the refinement factor is about 0.1 μg / ml to about 10 μg / ml (e.g., about 0.2 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml to about 2.5 μg / ml, e.g., about 1 μg / ml).

[0199] In some embodiments, the concentration of CD40L in the refinement factor is about 1 μg / ml to about 100 μg / ml (e.g., about 2 μg / ml to about 50 μg / ml, e.g., about 5 μg / ml to about 20 μg / ml, e.g., about 10 μg / ml).

[0200] In some embodiments, the concentration of the anti-PD-L1 antibody in the refinement factor is from about 1 μg / ml to about 200 μg / ml (e.g., from about 5 μg / ml to about 100 μg / ml, e.g., from about 10 μg / ml to about 50 μg / ml, e.g., about 20 μg / ml).

[0201] In some embodiments, the concentration of TPI-1 in the refining agent is about 0.1 μg / ml to about 10 μg / ml (e.g., about 0.2 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml to about 2.5 μg / ml, e.g., about 1 μg / ml).

[0202] In some embodiments, the concentration of R848 in the refining agent is about 0.1 μg / ml to about 10 μg / ml (e.g., about 0.2 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml to about 2.5 μg / ml, e.g., about 1 μg / ml).

[0203] Methods for promoting survival of monocytes - Patent application The present application provides various methods for promoting monocyte survival. In some embodiments, monocytes are obtained (e.g., freshly isolated) from an individual (e.g., a human). In some embodiments, the individual has cancer (e.g., any type or type of cancer described herein). In some embodiments, the individual has a disease or condition associated with immunosuppression (e.g., fibrosis, post-organ transplant under immunosuppressive drugs). In some embodiments, the individual has a viral infection. In some embodiments, monocytes obtained from an individual express lower levels of IL-10 receptor ("IL-10R"), IL-4 receptor ("IL-4R"), IL-6 receptor ("IL-6R"), M-CSF receptor ("GM-CSFR") and / or M-CSF receptor ("GM-CSFR") compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0204] In some embodiments, the present application provides a method of promoting survival of a monocyte population from an individual in in vitro culture, comprising culturing the monocyte population in a medium having an IL-10R activator, optionally wherein the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the population of monocytes expresses a low level (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) of IL-10R prior to contact with the IL-10R activator, compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the culture further comprises a TNFα receptor (TNFR) activator and / or an interferon gamma (IFNγ) receptor (IFNGR) activator, optionally wherein the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, optionally wherein the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally wherein the culture further comprises TNFα and / or IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml).In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0205] In some embodiments, the present application provides a method of promoting survival of a monocyte population from an individual in in vitro culture, comprising culturing the monocyte population in a medium having a TNFα receptor (TNFR) activator and / or an interferon gamma (IFNγ) receptor (IFNGR) activator, optionally wherein the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, optionally wherein the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally wherein the culture comprises TNFα and / or IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml). In some embodiments, the population of monocytes expresses a low level (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) of IL-10R prior to contact with the IL-10R activator, compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0206] In some embodiments, the present application provides a method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium having IL-10, TNFα, and IFNγ. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0207] In some embodiments, the culture further comprises a GM-CSF receptor (GM-CSFR) activator. In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml).

[0208] In some embodiments, the culture further comprises an IL-6 receptor (IL-6R) activator. In some embodiments, the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R. In some embodiments, the IL-6R activator is IL-6. In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally from about 5 pg / ml to about 100 pg / ml (e.g., from about 10 to 50 pg / ml, e.g., about 30 pg / ml).

[0209] In some embodiments, the present application provides a method of promoting survival of a monocyte population from an individual in in vitro culture, comprising culturing the monocyte population in medium derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies, the medium comprising an activator of IL-10R. In some embodiments, the T cells are CD4 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the medium is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally for about 2 days. In some embodiments, the population of monocytes expresses a lower level (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) of IL-10R prior to contact with the IL-10R activator compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0210] Anti-CD3 / CD28 treatment of T cells as described herein is a technique well known in the art for activating T cells.

[0211] The application further provides a method of increasing expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual with cancer, comprising contacting the population of monocytes with one or more agents selected from the group consisting of an IL-10R activator, a TNFR activator, and an IFNGR activator. In some embodiments, the population of monocytes expresses a lower level (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) of IL-10R prior to contact with the IL-10R activator, compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). In some embodiments, the IL-10R activator is IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the population of monocytes expresses a low level (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) of IL-10R prior to contact with the IL-10R activator, compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR. In some embodiments, the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.In some embodiments, the IFNGR activator comprises TNFα. In some embodiments, the IFNGR activator comprises IFNγ. In some embodiments, IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0212] In some embodiments, the monocytes are cultured for at least about 2 days (eg, about 2-3 days).

[0213] Methods for promoting differentiation of a population of monocytes into antigen-presenting cells ("APCs") The present application provides a method of promoting differentiation of a population of monocytes from an individual (e.g., a cancer patient or a patient with a viral infection) into antigen presenting cells ("APCs") in in vitro culture, the method comprising culturing the population of monocytes in a medium having one or more molecules selected from the group consisting of an IL-4 receptor (IL-4R) activator (e.g., IL-4), a TNFα receptor (TNFR) activator (e.g., TNFα) and an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), optionally wherein the monocytes are contacted with an IL-10 receptor (IL-10R) activator (e.g., IL-10) or the medium further comprises an IL-10 receptor (IL-10R) activator. In some embodiments, a method is provided for promoting differentiation of a population of monocytes from an individual (e.g., a cancer patient) into antigen-presenting cells ("APCs") in in vitro culture, comprising culturing the population of monocytes in a medium having an IL-4 receptor (IL-4R) activator (e.g., IL-4), a TNFα receptor (TNFR) activator (e.g., TNFα), and an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ). In some embodiments, the IL-4R activator is selected from the group consisting of IL-4, IL-13, an IL-4R agonist antibody, and a small molecule activator of IL-4R. In some embodiments, the IL-4R activator is IL-4. In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml). In some embodiments, the TNFR activator is selected from the group consisting of TNFα, TNFR agonist antibodies, and small molecule activators of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the TNFα is human TNFα or human recombinant TNFα.In some embodiments, TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR. In some embodiments, the IFNGR activator is IFNγ. In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the culture further comprises an IL-6 receptor (IL-6R) activator. In some embodiments, the IL-6R activator is selected from the group consisting of IL-6, IL-6R agonist antibodies, and small molecule activators of IL-6R. In some embodiments, the IL-6R activator is IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally from about 5 pg / ml to about 100 pg / ml (e.g., from about 10 to 50 pg / ml, e.g., about 30 pg / ml). In some embodiments, the monocytes obtained from the individual express lower (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) levels of IL-4 receptor ("IL-4R") compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0214] In some embodiments, a method is provided for promoting differentiation of a population of monocytes from an individual (e.g., a cancer patient) into antigen presenting cells ("APCs") in in vitro culture, comprising culturing the population of monocytes in a medium having IL-4, TNFα, and IFNγ. In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the monocytes obtained from the individual express lower (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) levels of IL-4 receptor ("IL-4R") compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0215] In some embodiments, a method is provided for promoting differentiation of a population of monocytes from an individual (e.g., a cancer patient) into antigen presenting cells ("APCs") in in vitro culture, comprising culturing the population of monocytes in a medium having IL-4, IL-6, TNFα, and IFNγ, and optionally wherein the monocytes have a lower (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower) level of IL-4 receptor (IL-4R) expression compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml). In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally at about 0.5 ng / ml to about 30 ng / ml (e.g., about 1 to 10 ng / ml). In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml). In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally from about 5 pg / ml to about 100 pg / ml (e.g., from about 10 to 50 pg / ml, e.g., about 30 pg / ml).

[0216] In some embodiments, the culture further comprises a GM-CSF receptor (GM-CSFR) activator. In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml).

[0217] In some embodiments, the culture further comprises an IL-10 receptor (IL-10R) activator. In some embodiments, the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). In some embodiments, the IL-10R activator is IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, the IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml).

[0218] In some embodiments, the monocytes are cultured for at least about 2 days (eg, about 2-3 days).

[0219] Method for obtaining tumor-specific or virus-specific antigen-presenting cells In some embodiments, a method of obtaining tumor-specific antigen presenting cells is provided, comprising: a) separately or simultaneously contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs; and b) contacting the APCs with a composition comprising a tumor antigen (e.g., a plurality of synthetic tumor antigen peptides), thereby obtaining said tumor-specific APCs.

[0220] In some embodiments, the composition comprising a tumor antigen comprises tumor cells (eg, a tumor biopsy or cells from a patient, such as a fresh or frozen-thawed tumor biopsy sample, eg, cultured tumor cells from a tumor biopsy).

[0221] In some embodiments, the composition comprising a tumor antigen comprises a neo-antigen peptide (e.g., a personalized neo-antigen long peptide (LP)). In some embodiments, the neo-antigen peptide is AI-identified and synthesized. See, e.g., Mor SK et al. Oncoimmunology. 2022 Jan 10;11(1):2023255.

[0222] In some embodiments, the composition comprising a tumor antigen comprises a shared tumor-associated antigen. In some embodiments, the shared tumor-associated antigen comprises an autoantigen (e.g., an aberrantly expressed autoantigen). In some embodiments, the autoantigen is selected from the group consisting of melanoma antigen-1 (MAGE-1), prostate-associated PAP, PSA and PSMA, breast cancer-associated BCAR3, and multi-cancer-associated MUC1. In some embodiments, the shared tumor-associated antigen comprises a non-autoantigen of viral origin (e.g., an antigen derived from LMP1 / 2 associated with nasopharyngeal carcinoma and lymphoma, e.g., an antigen derived from E6 and E7 proteins of high-risk human papillomavirus (HPV), e.g., an antigen derived from a retroviral Tax protein found in adult T-cell leukemia). In some embodiments, the shared tumor-associated antigen comprises a mutation-causing neoantigen shared by different types of cancer (e.g., a neoantigen associated with p53 mutation or KRAS mutation).

[0223] In some embodiments, the tumor antigen peptide (e.g., synthetic tumor antigen peptide) is obtained by a) identifying a tumor-specific mutation in a tumor tissue sample of a patient with a virus-associated cancer, where the tumor-specific mutation is not present in the virus, and b) synthesizing a peptide that includes the tumor-specific mutation. In some embodiments, the tumor-specific mutation is identified by sequencing the tumor tissue sample and the virus sample and comparing the sequences from the two samples.

[0224] In some embodiments, the tumor antigen peptide (e.g., synthetic tumor antigen peptide) is obtained by a) identifying a tumor-specific mutation in a tumor tissue sample of a patient with cancer that is not present in a normal tissue sample of the cancer patient, and b) synthesizing a peptide that includes the tumor-specific mutation. In some embodiments, the tumor-specific mutation is identified by sequencing the tumor tissue sample and the normal tissue sample and comparing the sequences from the two samples.

[0225] In some embodiments, a method of obtaining tumor-specific antigen-presenting cells is provided, comprising: a) separately or simultaneously contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs; and b) introducing a polynucleotide encoding a tumor antigen (such as any tumor antigen described herein) into the population of APCs. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is mRNA.

[0226] In some embodiments, a method of obtaining virus-specific antigen presenting cells is provided, comprising: a) separately or simultaneously contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs; and b) contacting the APCs with a composition comprising a viral antigen, thereby obtaining virus-specific APCs. In some embodiments, the composition comprising a viral antigen comprises a viral sample.

[0227] In some embodiments, a method of obtaining virus-specific antigen-presenting cells is provided, comprising: a) separately or simultaneously contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs; and b) introducing a polynucleotide encoding a viral antigen into the population of APCs. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is mRNA.

[0228] In some embodiments, a population of tumor-specific or virus-specific APCs produced by any of the methods described herein is provided.

[0229] Monocytes The methods described herein convert a plurality of monocytes into APCs. In some embodiments, the plurality of monocytes is obtained from the peripheral blood of an individual.

[0230] In some embodiments, monocytes express CD14 when obtained from peripheral blood.Methods for obtaining monocytes from peripheral blood are well known in the art.For example, PBMCs can be plated on cell culture dishes to allow monocyte adhesion, which is a common method for separating monocytes from non-adherent lymphocytes.Monocytes can also be separated by positive selection with anti-CD14 Ab or negative selection using all Abs against other cells.

[0231] In some embodiments, monocytes obtained from the individual express lower levels of IL-10 receptor ("IL-10R") prior to contact with one or more of the cytokines listed above, compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the level of IL-10R on monocytes from the individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-10R on monocytes from a reference individual (e.g., a healthy individual).

[0232] In some embodiments, monocytes obtained from the individual express lower levels of IL-4 receptor ("IL-4R") prior to contact with one or more cytokines listed above, compared to monocytes obtained from a healthy individual. In some embodiments, the level of IL-4R on monocytes from the individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0233] In some embodiments, monocytes obtained from an individual express lower levels of IL-6 receptor ("IL-6R") prior to contact with one or more cytokines as described above, compared to monocytes obtained from a healthy individual. In some embodiments, the level of IL-6R on monocytes from an individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% lower than the level of IL-6R on monocytes from a reference individual (e.g., a healthy individual).

[0234] In some embodiments, monocytes obtained from an individual express lower levels of M-CSF receptor ("M-CSFR") prior to contact with one or more cytokines listed above, compared to monocytes obtained from a healthy individual. In some embodiments, the level of M-CSFR on monocytes from an individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% lower than the level of M-CSFR on monocytes from a reference individual (e.g., a healthy individual).

[0235] In some embodiments, monocytes obtained from an individual express lower levels of GM-CSF receptor ("GM-CSFR") prior to contact with one or more cytokines listed above, compared to monocytes obtained from a healthy individual. In some embodiments, the level of GM-CSFR on monocytes from an individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% lower than the level of GM-CSFR on monocytes from a reference individual (e.g., a healthy individual).

[0236] In some embodiments, the methods described herein further include assessing IL-10R expression levels in monocytes (e.g., prior to contacting the monocytes with one or more cytokines, such as IL-10, IFNγ and / or TNFα).

[0237] cancer The cancers described in this section (e.g., in relation to monocytes derived from cancer patients) can be of any type or variety. All cancer types discussed in Section V are applicable here as well.

[0238] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0239] In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a late stage cancer. In some embodiments, the cancer is in stage II, III or IV. In some embodiments, the cancer is an inoperable tumor and / or is malignant.

[0240] Examples of cancers described herein include adrenocortical carcinoma, primary myelofibrosis, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and and hypothalamic glioma, glioblastoma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary site, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic neoplasia, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatocellular carcinoma) and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g. small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), lymphatic tumors (e.g. lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndromes, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine carcinoma, oropharyngeal cancer, ovarian cancer (e.g. ovarian epithelial carcinoma, ovarian germ cell tumors, ovarian low malignant tumors, large-scale tumors), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g. non-melanoma (e.g. squamous cell carcinoma), melanoma, Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor,These include, but are not limited to, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0241] In some embodiments, the cancer is a viral infection-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cervical cancer, e.g., HPV-associated head and neck cancer, e.g., HPV-associated squamous cell carcinoma). In some embodiments, the cancer is a human herpesvirus 8 (HHV8)-associated cancer (e.g., Kaposi's sarcoma). In some embodiments, the cancer is a human T-lymphoproliferative virus (HTLV-1)-associated cancer (e.g., adult T-cell leukemia or lymphoma). In some embodiments, the cancer is an Epstein-Barr virus (EBV)-associated cancer (e.g., Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma, gastric cancer). In some embodiments, the cancer is a hepatitis B virus (HBV)-associated cancer (e.g., liver cancer). In some embodiments, the cancer is a hepatitis C virus)-associated cancer (e.g., liver cancer, non-Hodgkin's lymphoma).

[0242] In some embodiments, the cancer is refractory to one or more radiation therapies, chemotherapy, or immunotherapy (eg, checkpoint blockade).

[0243] In some embodiments, the cancer is liver cancer, renal cancer, endometrial cancer, thymic epithelial neoplasm, lung cancer, spindle cell sarcoma, chondrosarcoma, uterine smooth muscle, pancreatic cancer.

[0244] individual In some embodiments, the individual is healthy or does not exhibit symptoms of a disease or condition (e.g., cancer). In some embodiments, the individual has cancer or a tumor. In some embodiments, the individual has a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0245] In some embodiments, the individual has advanced cancer. In some embodiments, the individual has late stage cancer. In some embodiments, the individual has stage II, III or IV cancer. In some embodiments, the individual has inoperable tumors and / or metastases. In some embodiments, the cancer is malignant.

[0246] In some embodiments, the individual has an infection (eg, a viral infection, a bacterial infection).

[0247] In some embodiments, the individual is female. In some embodiments, the individual is male.

[0248] In some embodiments, the individual is a human. In some embodiments, the individual is a human who is at least about 50, 55, 60, 65, 70, or 75 years of age.

[0249] III. Antigen presenting cells (APCs), compositions and cultures. The present application provides APCs, such as those prepared according to any of the above methods, with unique properties that distinguish them from natural APCs or APCs generated in vitro by currently known methods. Comprehensive studies, discussed in detail in the Examples, show that the exemplary APCs of the present application differ from, for example, dendritic cells (e.g., cDC1, cDC2, pDC) or macrophages (e.g., M1 macrophages, M2 macrophages) in shape / size, the way they adhere to matrix substratum, various cell surface molecules, antigen presenting capacity, and / or gene transcription profile. See, for example, Figures 14-19C. The results as a whole suggest that the exemplary APCs of the present application are a unique cell population that are generally smaller (approximately 7-15 μm after trypsinization) than macrophages / dendritic cells (approximately 10-20 μm) with a different shape, but have superior antigen presenting capacity and are highly sensitive to various types of antigens, as evidenced, for example, by their high expression of CD40, TLR2 and STING. The exemplified APCs inherently express high levels of LOX1 and uPAR and have a significantly different gene transcription profile compared to mature DCs or macrophages. These data demonstrate that the APCs of the present application represent a novel and robust APC population that is fundamentally different from currently known APCs.

[0250] The exemplary APCs described herein have surface markers that are distinct from known dendritic cell subsets, such as myeloid cDC1, myeloid cDC2, plasmacytoid DC (pDC) or Mo-DC, and known human macrophages (such as M1 and M2 macrophages) or monocytes. See, for example, Figures 18A-18B, 19A-19C and 15A-15B. For example, cDC1 expresses high levels of CD141, CLEC9A, XCR1, CD103, CD103 and DEC205, while the exemplary APCs of the present application do not express or express much lower levels than all of the above cDC1 markers. Similarly, cDC2 expresses high levels of SIPRa and CD1c, while the exemplary APCs of the present application express much lower levels of both of them. pDC expresses high levels of CD303, while the exemplary APCs of the present application express much lower levels than CD303. MoDCs express high levels of CD205, SIRPa, CD1c, CD11c, CD303, CD209, whereas the exemplary APCs of the present application express none or much lower levels of any of these surface markers. M1 macrophages express high levels of CD26, SIRPa, CD11c, CCR2, CCR7, CD14, CD303, PD-L1, CLEC5, CCR1, whereas the exemplary APCs of the present application have similar expression of CD14 and PD-L1 but different expression of all other surface markers.

[0251] On the other hand, the exemplary APCs of the present application consistently express several surface molecules that are not expressed or are expressed at relatively low levels in the above-mentioned dendritic cells or macrophages. These molecules include the receptor for oxidized LDL (LOX1), the receptor for urokinase plasminogen activator (uPAR), the receptor for IL-3 (IL-3R) and the receptor for complement component 3a (C3AR), TLR2 and / or STING. Neither dendritic cells nor macrophages have a similar expression pattern of these molecules.

[0252] Consistently, the exemplified APCs of the present application have different morphologies, for example, as shown in FIG. 14. These APCs also show different gene transcription profiles, as shown in FIG. 16A-16C. As shown, these cells are smaller than dendritic cells or macrophages and have different cell shapes. These exemplary APCs have unique patterns of gene transcription profiles from dendritic cells, macrophages or monocytes, but APCs a) derived from monocytes from cancer patients, b) derived from monocytes from healthy individuals, c) cultured in KX1, and d) cultured in c-combo show very similar gene transcription profiles. These results again demonstrated that the APCs of the present application are unique and non-transient APC populations.

[0253] In some embodiments, a population of APCs is provided that a) are MHC-I+ / high and MHC-II+ / high, b) express or express high levels of at least one or more (e.g., 2, 3, 4) costimulatory molecules including CD40, CD80 and CD86, and / or OX40L+ / high, and / or PD-L1+ / high, c) TLR2+ and / or STING+, d) do not express or express at least one or more (e.g., 2, 3, 4, 5, 6) cDC1 surface molecules including CD141, Clec9a, CD26, XCR1, CD103, DEC205, compared to cDC1, and e) are not expressed or express at least one or more (e.g., 2, 3, 4, 5, 6) cDC1 surface molecules including CD141, Clec9a, CD26, XCR1, CD103, DEC205, compared to cDC2. f) express lower levels of at least one (e.g., 2, 3, 4, or 5) dendritic cell or macrophage surface molecule, including CD11c, CCR2, CCR7, CD14, and CD303, compared to MoDCs (e.g., derived from monocytes following treatment with LPS or TNFα); g) express lower levels of the pDC surface molecule CD303; and / or h) express high levels of uPAR and / or LOX1 (e.g., express higher levels of LOX1 and / or uPAR compared to monocytes, LPS-MoDCs, or M1 / M2 macrophages). In some embodiments, the APCs are OX40L+ / high, ICOSL+, CD70+, and / or 4-1BBL+, or have increased expression of OX40L, ICOSL, CD70, and / or 4-1BBL, compared to monocytes (e.g., the monocytes from which they are derived). In some embodiments, the APCs are CD31+ / low or have decreased CD31 compared to monocytes (e.g., the monocytes from which they are derived). In some embodiments, the APCs are PD-L1+ / high or have increased PD-L1 compared to monocytes (e.g., the monocytes from which they are derived). In some embodiments, the APCs are SIRPa- / low, LilRB- / low, and / or Siglec- / low or have decreased expression of SIRPa, LilRB, and Siglec compared to monocytes (e.g., the monocytes from which they are derived).In some embodiments, the APCs are CD32+ / high, Trem2high, IL-3Rhigh, and / or c-Met+ / high. In some embodiments, the APCs are TLR3+ / high and / or TLR8+ / high (e.g., have higher TLR3 and / or TLR8 expression than M1 macrophages). In some embodiments, the APCs express lower levels of CD14 compared to monocytes. In some embodiments, the APCs have a size of less than about 15 μm (diameter) (e.g., about 7-15 μm). In some embodiments, the APCs have multiple shapes (multi-shapes) when attached to a matrix and can elongate / stretch to assume a spindle shape. After trypsinization, these cells become round and have a uniform size of less than about 15 μm (e.g., about 7-15 μm).

[0254] As described herein, "+ / high" refers to positive expression of a particular surface molecule, or high expression of a particular surface molecule. In some embodiments, high expression refers to a scenario in which a cell (e.g., APC) expresses a higher level of a surface molecule than a reference cell population. In some embodiments, the reference cell population is a monocyte, macrophage (e.g., M1 macrophage or M2 macrophage), dendritic cell (e.g., Mo-DC, cDC1, cDC2, pDC) that is known to express this surface molecule. In some embodiments, higher level refers to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, or 100-fold higher expression level. In some embodiments, increased expression of a particular molecule refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, or 100-fold higher. In some embodiments, decreased expression of a particular molecule refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% lower.

[0255] In some embodiments, a population of APCs is provided, wherein the APCs are LOX1+ / high, uPAR+ / high, CD40+ / high, and / or TLR2+ / high. In some embodiments, the APCs have a CD40 surface expression level that is at least 5-fold or 10-fold higher than dendritic cells (e.g., dendritic cells derived from monocytes following GM-CSF / IL-4 treatment as described herein followed by LPS treatment) or M1 / M2 macrophages (e.g., M1 / M2 macrophages derived from monocytes following M-CSF treatment followed by LPS and IFNγ treatment for an M1 phenotype, or LPS, IL4 and IL-10 treatment for an M2 phenotype). In some embodiments, the APCs have comparable or higher surface expression of MHC-I, MHC-II, CD80, CD86, and CD40 compared to dendritic cells (e.g., dendritic cells derived from monocytes after GM-CSF / IL-4 treatment followed by LPS treatment as described herein) or M1 / M2 macrophages (e.g., M1 / M2 macrophages derived from monocytes after M-CSF treatment followed by LPS and IFNγ treatment for an M1 phenotype, or LPS, IL4, and IL-10 treatment for an M2 phenotype). In some embodiments, the APCs are IL-3R+ / high, TREM2+ / high, C3AR+ / high, and PD-L1+ / high. In some embodiments, the APCs have a size of less than about 15 μm (diameter) (e.g., about 7-15 μm). In some embodiments, the APCs have multiple shapes (multi-shapes) when attached to a matrix and can elongate / extend to assume a spindle shape. After trypsinization, the cells become rounded and have a uniform size of less than about 15 µm (e.g., between about 7 and 15 µm).

[0256] In some embodiments, a population of APCs (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient) is provided, where the APCs are a) MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), and b) have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APCs have multiple shapes (multi-shape) when attached to a matrix and can elongate / stretch to assume a spindle shape. After trypsinization, these cells become round and have a uniform size of less than about 15 μm (e.g., about 7-15 μm). They are smaller than both dendritic cells and macrophages. See, e.g., FIG. 14. In some embodiments, they are TLR2+ / high and STING+ / high (e.g., express higher levels of TLR2 and STING than M1 macrophages), and / or express higher levels of CD40 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs. In some embodiments, APCs a) do not express or express low levels of CD141, XCR1, and CD103 compared to cDC1s, b) do not express or express low levels of CD1c and SIRPa compared to cDC2s, c) express low levels of CD303 compared to pDCs or M1 macrophages, and d) express low levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, APCs have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, APCs have a spindle or elongated shape, or polymorphism. In some embodiments, APCs adhere to the matrix substratum but can be easily dislodged by repeated pipetting or brief trypsinization (<2 min at 37° C.). In some embodiments, APCs are a) CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages and MoDCs).In some embodiments, the expression level of CD40 on APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0257] In some embodiments, a population of APCs (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient)) is provided, the APCs being 1) MHC-I+ / high and MHC-II+ / high (e.g., expressing higher levels of MHC-I and MHC-II compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), and 2) a) expressing or not expressing low levels of CD141, XCR1, and CD103 compared to cDC1, b) expressing low levels of CD1c and SIRPa compared to cDC2, c) expressing low levels of CD303 compared to pDC or M1 macrophages, and / or d) expressing low levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, the APCs have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APCs are a) CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs). In some embodiments, the APCs express similar or higher levels of CD80, CD86, CD40, OX40L, ICOSL, and / or CD70 compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα). In some embodiments, the APCs express higher levels of LOX1, uPAR, CD40, TLR2, IL-3R, TREM2, C3AR, IL-3R, and / or PD-L1 compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα), monocytes, M1, or M2 macrophages.

[0258] In some embodiments, a population of APCs (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient)) is provided, where the APCs a) are MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), b) are TLR2+ / high and STING+ / high (e.g., express higher levels of TLR2 and STING than M1 macrophages), and c) express higher levels of CD40 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs. In some embodiments, APCs a) do not express or express low levels of CD141, XCR1, and CD103 compared to cDC1, b) do not express or express low levels of CD1c and SIRPa compared to cDC2, c) express low levels of CD303 compared to pDC or M1 macrophages, and d) express low levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, APCs have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, APCs have a spindle or elongated shape, or pleomorphism. In some embodiments, APCs adhere to the matrix substratum but can be easily dislodged by repeated pipetting or brief trypsinization (less than 2 min at 37 °C). In some embodiments, the APCs are a) CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs). In some embodiments, the expression level of CD40 on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0259] In some embodiments, a population of APCs (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient) is provided, where the APCs a) are MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), b) express or express high levels of at least one of LOX1 and uPAR (e.g., higher levels of LOX1 and uPAR compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), and c) express high levels of CD40 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs. In some embodiments, the APCs express high levels of at least one of TLR2 and STING (e.g., higher levels of TLR2 and STING than M1 macrophages). In some embodiments, APCs a) do not express or express low levels of CD141, XCR1, and CD103 compared to cDC1, b) do not express or express low levels of CD1c and SIRPa compared to cDC2, c) express low levels of CD303 compared to pDC or M1 macrophages, and d) express low levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, APCs have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, APCs have a spindle or elongated shape, or pleomorphism. In some embodiments, APCs adhere to the matrix substratum but can be easily dislodged by repeated pipetting or brief trypsinization (less than 2 min at 37 °C). In some embodiments, the APCs are a) CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs). In some embodiments, the expression level of CD40 on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0260] In some embodiments, a population of APCs (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient)) is provided, the APCs a) are MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs); b) express high levels of at least one of LOX1 and uPAR (e.g., higher levels of LOX1 and uPAR compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs); and c) are TLR2+ / high and STING+ / high (e.g., express higher levels of TLR2 and STING than M1 macrophages). In some embodiments, APCs a) do not express or express low levels of CD141, XCR1, and CD103 compared to cDC1, b) do not express or express low levels of CD1c and SIRPa compared to cDC2, c) express low levels of CD303 compared to pDC or M1 macrophages, and d) express low levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, APCs have a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, APCs have a spindle or elongated shape, or pleomorphism. In some embodiments, APCs adhere to the matrix substratum but can be easily dislodged by repeated pipetting or brief trypsinization (less than 2 min at 37 °C). In some embodiments, the APCs are a) CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs). In some embodiments, the expression level of CD40 on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0261] In some embodiments, a population of APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient) is provided, the APCs a) have a spindle-shaped or elongated shape or are polymorphic, b) express higher or similar levels of MHC-I, MHC-II, CD80, CD86, CD40, OX40L, ICOSL, and / or CD70 compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα), c) express higher levels of LOX1, uPAR, CD40, TLR2, IL-3R, TREM2, IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, IL-27R, IL-28R, IL-29 ... d) express lower levels of DEC205, SIRPa and / or CD11c compared to mature dendritic cells (e.g. derived from monocytes after culture with LPS or TNFα) or M1 macrophages; e) express low levels of CD11c and CD303 compared to mature dendritic cells (e.g. derived from monocytes after culture with LPS or TNFα) or M1 macrophages; f) express lower levels of CD26, CCR7 and / or CCR2 than M1 macrophages; and / or g) express high levels of Sting and / or TLR2, and optionally TLR3 and / or TLR8, compared to M1 macrophages.

[0262] In some embodiments, APCs express higher levels of CCL3L1, CXCL8, IL-6, IL1B, CCL2, CXCL1, CXCL2, CXCL3, CCL7, C3AR1, SLC16A6, CXCL5, and / or SERPINB2 compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS). In some embodiments, APCs express lower levels of CCL22, CSTB, LIPA, CCL17, CCL13, APOE, FABP4. CD1B, FN1, CD1c, CD1A, and / or PTGDS compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS). In some embodiments, APCs express higher levels of CXCL8, IL-6, NAMPT, CXCL1, CCL6, CXCL3, CCL18, PELI1, CXCL5, SLC16A6, and / or SERPINB2 compared to M1 macrophages. In some embodiments, APCs express lower levels of CXCL10, MMP9, LIPA, S100A4, CCL22, IL12B, APOE, CRABP2, PTGDS, and / or FN1 compared to M1 macrophages. In some embodiments, APCs express higher levels of C3AR1, olr1, TLR2, and PLAUR compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα), M1 / M2 macrophages, and / or monocytes. In some embodiments, APCs express lower levels of DC-specific antigens, such as CD11c, CD1a, CD1c, Batf3, compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα). In some embodiments, APCs express lower levels of macrophage-specific antigens (e.g., CD68) compared to M1 / M2 macrophages. In some embodiments, APCs express lower levels of monocyte-specific antigens (e.g., CCR2, CXCR1, CD14). In some embodiments, APCs express lower levels of CD31 compared to monocytes.

[0263] In some embodiments, a population of APCs derived from monocytes obtained from an individual (e.g., a cancer or virally infected patient) is provided, the APCs a) express high levels of one or more antigen presenting molecules, the antigen presenting molecules selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, and / or b) express low levels of inhibitory signaling molecules, the inhibitory signaling molecules selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2), and Siglec 10. In some embodiments, the monocytes exhibit lower expression levels of M-CSFR, GM-CSFR, IL-6R, IL-10R and / or IL-4R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the APCs have dendritic cell morphology.

[0264] In some embodiments, a population of APCs is provided that is derived, for example, from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), the APCs having: a) high levels of MHC I, MHC a) express high levels of IL-12, type I and / or type II IFN, TNFα, IL-1 and IL-6 (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the levels of the corresponding molecules on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF for about 2 days); and c) produce low levels of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglecs. 10 (e.g., at least about 10%, 20%, 30%, 40%, or 50% lower than the levels of the corresponding molecules on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF for about 2 days), and / or d) at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the APCs have dendritic cell morphology.

[0265] The APCs described herein are derived in some embodiments from monocytes obtained from an individual (e.g., a cancer patient or a viral infection patient). In some embodiments, the monocytes exhibit lower M-CSFR expression levels (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes exhibit lower GM-CSFR expression levels (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes exhibit lower expression levels of both M-CSFR and G-CSFR (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0266] In some embodiments, the monocytes exhibit a lower IL-10R expression level (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes exhibit a lower IL-6R expression level (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes exhibit a lower IL-4R expression level (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes exhibit a lower IFNGR expression level (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0267] In some embodiments, the APCs express high levels of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen-presenting molecules, the antigen-presenting molecules being selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, and optionally the APCs are generated from monocytes in cell culture (e.g., any monocytes described herein, e.g., monocytes obtained from a cancer patient). In some embodiments, the APCs express high levels of MHC I, MHC II, CD86, CD80, CD40, and / or OX40L.

[0268] In some embodiments, the APCs express high levels of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen-presenting molecules, wherein the level of one or more antigen-presenting molecules selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40 is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the level of the corresponding molecule on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF (e.g., for about 2 days).

[0269] In some embodiments, the APCs express high levels of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen-presenting molecules, where the level of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen-presenting molecules selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40 is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher than the level of the corresponding molecule on dendritic cells obtained from a healthy human and cultured with GM-CSF and IL-4 for about 5 days, where the antigen-presenting molecule is selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40.

[0270] In some embodiments, the APCs express high levels of MHC I, MHC II, CD86, CD80, CD40, and / or OX40L.

[0271] In some embodiments, the APCs produce high levels of one or more (e.g., at least 2, 3, 4, 5, or 6) cytokines selected from the group consisting of IL-12, type I and / or type II IFN, TNFα, IL-1, and IL-6 if the levels of the cytokines are at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the levels of the corresponding cytokines on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF (e.g., for about 2 days).

[0272] In some embodiments, APCs produce high levels of PD-L1 if the level of PD-L1 is at least about 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175% or 200% higher than the level of PD-L1 on monocytes from which they were derived when obtained from an individual.

[0273] In some embodiments, the APCs express low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10. In some embodiments, the APCs express low levels of SIRPα.

[0274] In some embodiments, APCs express low levels of inhibitory signaling molecules when the level of one or more antigen-presenting molecules selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10 is at least about 10%, 20%, 30%, 40%, or 50% lower than the level of the corresponding molecule on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF (e.g., for about 2 days).

[0275] In some embodiments, the APCs express low levels of inhibitory signaling molecules when the level of one or more antigen-presenting molecules selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10 is at least about 10%, 20%, 30%, 40%, or 50% lower than the level of the corresponding molecule on dendritic cells obtained from healthy humans and cultured with GM-CSF and IL-4 for about 5 days.

[0276] In some embodiments, the APCs have substantially the same morphology as that shown in FIG. 4F (right).

[0277] In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the APCs have dendritic cell morphology.

[0278] In some embodiments, the APC comprises one or more tumor-associated antigenic peptides, such as neo-antigenic peptides (such as any of those discussed herein).

[0279] In some embodiments, the APC comprises one or more virus-associated antigenic peptides.

[0280] In some embodiments, the APCs can promote the proliferation of immune cells (e.g., T cells, e.g., CD4 T cells and / or CD8 T cells) upon incubation with the immune cells. In some embodiments, the APCs promote the proliferation of T cells at least about 5-fold, 10-fold, 15-fold, or 20-fold in cultures containing IL-2, IL-7, and IL-15. In some embodiments, the incubation is for no more than about 24 hours, 22 hours, 20 hours, or 18 hours. In some embodiments, the APCs present one or more disease-associated peptides (e.g., tumor peptides) to the immune cells.

[0281] In some embodiments, compositions (e.g., cultures) are provided that include the APCs described herein. In some embodiments, the APCs present one or more disease-associated peptides (e.g., tumor peptides) to immune cells.

[0282] Tumor-associated antigen peptides Various approaches are available for identifying tumor-associated antigenic peptides.

[0283] One approach often used to identify peptides recognized by such CTLs is expression cloning, which consists of isolating the genes encoding the peptides by transfecting a library of tumor cDNAs and testing the transfected cells for their ability to activate CTL clones. Fragments of the identified genes are then transfected to define the regions encoding the antigenic peptides, and finally, candidate peptides with the appropriate HLA binding motifs are tested for their ability to sensitize target cells to lysis by CTLs. This approach has been used successfully to identify a large number of antigenic peptides.

[0284] Today, tumor-associated antigenic peptides are often identified using a "reverse immunology" approach, which consists of selecting peptides with appropriate HLA-binding motifs inside the protein of interest, e.g., a protein encoded by a mutated oncogene, or a gene selectively expressed or overexpressed by the tumor. Candidate peptides are synthesized and tested for HLA binding in vitro. The most efficient binders are pulsed on antigen-presenting cells that are used to stimulate T lymphocytes in vitro to induce CTL lines or clones that recognize peptide-pulsed target cells. A drawback of this approach is that the identified peptides may not be efficiently processed by the tumor. It is therefore essential to verify that the CTLs recognize tumor cells that naturally express the gene encoding the peptide. In addition, transfectants expressing normal levels of the gene or cells in which expression of the gene has been knocked down using si or shRNA should be tested.

[0285] A third approach to antigen identification is based on elution of antigenic peptides from MHC class I molecules immunopurified from the surface of tumor cells. Direct identification of the sequences of eluted peptides by mass spectrometry, although technically demanding, has proven useful to identify or confirm the relevance of peptides that have undergone post-translational modifications such as serine / threonine phosphorylation, glycosylation-dependent asparagine deamidation, or peptide splicing.

[0286] Using these various approaches, a large number of antigenic peptides that are recognized by anti-tumor CTL have been identified.These antigens are conveniently classified according to the expression pattern of their parent genes.A regularly updated database of these antigenic peptides that are effectively presented by tumor cells can be found on the website of http: / / www.cancerimmunity.org / .See Vigneron, Biomed Res Int.2015;2015:948501.

[0287] The present application provides APCs produced by any of the methods described herein.

[0288] Neo-antigen peptides Various methods are available for detecting and screening neoantigens. Sandwich immunoassays in miniaturized systems have been able to successfully identify tumor antigens in serum samples extracted from patients. See, for example, Pollard et al., Proteomics Clin. Appl. 1 934-952 (2007); Yang et al., Biosens. Bioelectron. 40 385-392 (2013). Another tool, called serological proteome analysis (SERPA) or 2-D Western blot, consists of an isoelectric focusing (IEF) gel run in the first dimension and an SDS-PAGE gel run in the second dimension. SERPA separates proteins in the gel by their isoelectric point (IP) and molecular weight, then transfers the proteins from the gel to a carrier membrane for screening antibodies. Finally, antigen protein spots can be identified by MS. See, for example, Tjalsma et al., Proteomics Clin. Appl. 2 167-180 (2008). This approach has been used to identify antigens in different tumor types. Serological analysis of recombinant cDNA expression libraries (SEREX), which combines serological analysis and antigen cloning techniques, is a widely used technique to explore the antigen repertoire of tumors. SEREX first constructs a cDNA library from cancer cell lines or fresh tumor samples, then screens the cDNA library with autologous serum from cancer patients, and finally sequences the immunoreactive clones. SEREX has identified various tumor antigens, including CTAs, differentiation antigens, mutant antigens, splice variant antigens, and overexpressed antigens. See, for example, Chen et al., Proc. Natl. Acad. Sci. USA 94 1914-1918 (1997). In addition, other methods such as multiplex affinity protein profiling (MAPPing) and nanoplasmonic biosensors have also been developed to identify tumor antigens. See, for example, Lee et al., Biosens. Bioelectron. 74 341-346 (2015).

[0289] In some embodiments, one or more neo-antigen peptides described herein are obtained from a neo-antigen database (such as any of the neo-antigen databases described herein). For example, Tan et al. constructed a manually curated database for human tumor neo-antigen peptides ("dbPepNeo") based on the following four criteria: (i) the peptides are isolated from human tumor tissues or cell lines, (ii) the peptides contain non-synonymous mutations in the amino acid sequence, (iii) the peptides can bind to HLA-I molecules, and (iv) the peptides can induce CD8+ T cell responses. See Tan et al., Database (Oxford). 2020 Jan 1; 2020:baaa004. Xia et al. constructed another database, NEPdb, that provides pan-cancer-level predicted HLA-I neo-epitopes derived from 16,745 shared cancer somatic mutations using state-of-the-art predictors. See Xia et al., Front Immunol. 2021; 12:644637. Wu et al. developed a comprehensive tumor-specific neo-antigen database (TSNAdb v1.0) based on pan-cancer immunogenomic analysis of somatic mutation data and human leukocyte antigen (HLA) allele information for 16 tumor types using 7748 tumor samples from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). See Wu et al., Enomics Proteomics Bioinformatics. 2018 Aug;16(4):276-282.

[0290] In some embodiments, the one or more neo-antigenic peptides are obtained from analyzing biological information of an individual (such as a patient who had cancer). In some embodiments, the neo-antigenic peptides are obtained from a computational analysis of a cancer patient's tumor genome. See, e.g., Roudko et al. Front Immunol. 2020; 11:27. In some embodiments, the neo-antigenic peptides are obtained from a computational analysis of a cancer patient's transcriptome. See, e.g., Caushi et al., Nature. 2021 Aug; 596(7870): 126-132. In some embodiments, the neo-antigenic peptides are obtained from a computational analysis of a cancer patient's proteome. See, e.g., Wen et al., Nat Commun. 2020 Apr 9; 11(1): 1759.

[0291] In some embodiments, the neo-antigenic peptides are selected based on patient data. In some embodiments, the patient data is obtained from data from a group of patients with a particular type of cancer (e.g., any of the cancers described herein). In some embodiments, the patient data is derived from data from a group of patients with any cancer. In some embodiments, the patient group is of the same sex (e.g., male or female). In some embodiments, the patient group is of the same ethnicity. In some embodiments, the patient group has one or more biomarkers (e.g., abnormalities in a particular gene, e.g., KRAS, e.g., PTEN).

[0292] In some embodiments, one or more neo-antigenic peptides are derived from any polypeptide known or known to contain tumor-specific mutations. Suitable polypeptides from which neo-antigenic peptides can be derived can be found, for example, in various databases available in the art (e.g., COSMIC database). These databases manage comprehensive information on somatic mutations in human cancers. In some embodiments, the peptides comprise tumor-specific mutations. In some embodiments, the tumor-specific mutations are driver mutations for a particular cancer type.

[0293] In some embodiments, the tumor-associated peptides (e.g., neo-antigenic peptides) are synthetic peptides. In some embodiments, the neo-antigenic peptides are obtained by exome high-throughput sequencing and pre-screened with epitope prediction algorithms.

[0294] In some embodiments, one or more neo-antigenic peptides are selected based on their binding affinity to an MHC molecule (e.g., an MHC I molecule and / or an MHC II molecule). In some embodiments, the neo-antigenic peptide has a binding affinity (IC50) of less than 5000 nM (e.g., less than 500 nM, less than 250 nM, less than 100 nM, or less than 50 nM) to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity (IC50) of about 500 nM to 5000 nM to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity of less than 500 nM (IC50) to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity of about 250 nM to 500 nM to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity of less than 250 nM (IC50) to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity of less than 100 nM (IC50) to an MHC molecule. In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule with an IC50 of about 50 nM to 500 nM. In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule of less than 50 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule with an IC50 of about 1 nM to 50 nM.

[0295] In some embodiments, the multiple tumor-associated peptides (e.g., neo-antigenic peptides) are prepared from surgical resection of tumor tissue or a biopsy extract thereof.

[0296] In some embodiments, the multiple tumor-associated peptides (e.g., neo-antigenic peptides) are prepared from a mixture of tumor cells or extracts thereof isolated from tumor tissue or biopsy.

[0297] In some embodiments, the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) is prepared from a mixture of isolated tumor-associated peptides (e.g., neo-antigenic peptides).

[0298] In some embodiments, the tumor tissue or cells are fresh tumor tissue or cells. In some embodiments, the tumor tissue or cells are obtained from a frozen sample.

[0299] In some embodiments, tumor tissue or cells have been subjected to induction of immunogenic cell death (eg, freeze-thawing to lyse tumor cells, high dose UV irradiation, X-ray irradiation).

[0300] In some embodiments, the tumor tissue or cells have been subjected to radiation treatment.

[0301] IV. METHODS OF ACTIVATING IMMUNE CELLS AND ACTIVATED IMMUNE CELL COMPOSITIONS The present application also provides a method for activating a population of immune cells. In some embodiments, the method comprises co-culturing a population of immune cells with a population of APCs as described herein, wherein the APCs are pre-loaded with one or more antigenic peptides (e.g., tumor peptides, e.g., tumor-associated peptides, e.g., neo-antigenic peptides).

[0302] In some embodiments, a method of activating a population of immune cells (e.g., T cells, e.g., TIL cells) obtained from an individual (e.g., a cancer patient) is provided, comprising co-culturing the population of immune cells with a population of APCs (e.g., APCs described herein), thereby producing a population of activated immune cells, where the APCs are pre-loaded with one or more tumor peptides. In some embodiments, the APCs are derived from monocytes obtained from the same individual. In some embodiments, the APCs are pre-incubated with tumor antigens (e.g., free thawed tumor cells / debris). In some embodiments, the pre-incubation is for about 3 to 10 hours (e.g., about 6 hours). In some embodiments, the ratio of APCs to immune cells (e.g., T cells, e.g., TIL cells) in the co-culture is about 10:1 to about 1:10 (e.g., about 5:1 to about 1:5, about 2:1 to about 1:2, about 1:1). In some embodiments, the APCs and immune cells are co-cultured for about 2 to 20 days. In some embodiments, IL-2, IL-7 and / or IL-15 are supplemented to the co-culture (e.g., supplemented at least 2 or 3 days after co-culture). In some embodiments, the activated immune cells include at least 5, 10 or 20 times (e.g., 50-100 times) more cells than the immune cells before co-culture. In some embodiments, the activated immune cells are subjected to at least 2, 3, 4 or 5 rounds of activation via co-culture with APCs as described herein. In some embodiments, the activated immune cells do not exhibit exhaustive characteristics (e.g., senescence) after 2, 3 or 4 consecutive (e.g., about 6-10 days each) rounds of activation involving co-culture as described herein. In some embodiments, the co-culture does not include the use of anti-CD3 and / or anti-CD28 antibodies at least some times (e.g., anti-CD3 and anti-CD28 antibodies are used only in the first round and not in later rounds).

[0303] In some embodiments, the method includes contacting the APC with a composition comprising a plurality of tumor associated peptides (e.g., neo-antigenic peptides). In some embodiments, the APC is contacted with the composition comprising a plurality of tumor associated peptides (e.g., neo-antigenic peptides) for about 4 to about 24 hours.

[0304] In some embodiments, the APCs are pre-incubated with a composition comprising multiple tumor-associated peptides (eg, neo-antigenic peptides).

[0305] In some embodiments, the composition comprising a plurality of tumor-associated peptides (eg, neo-antigenic peptides) is a surgical resection of tumor tissue or a biopsy extract thereof.

[0306] In some embodiments, the composition comprising multiple tumor-associated peptides (e.g., neo-antigenic peptides) is a mixture of tumor cells or extracts thereof isolated from tumor tissue or biopsy.

[0307] In some embodiments, the composition comprising a plurality of tumor-associated peptides (e.g., neo-antigenic peptides) is a mixture of isolated tumor-associated peptides (e.g., neo-antigenic peptides).

[0308] In some embodiments, the tumor tissue or cells are fresh tumor tissue or cells, hi some embodiments, the tumor tissue or cells are obtained from a frozen sample.

[0309] In some embodiments, the tumor tissue or cells have been subjected to apoptosis induction.

[0310] In some embodiments, the tumor tissue or cells have been subjected to radiation treatment.

[0311] In some embodiments, the population of immune cells and the APCs are derived from the same individual.

[0312] In some embodiments, the population of immune cells and the antigen presenting cells are not derived from the same individual.

[0313] The application also provides activated immune cells (eg, T cells) produced by any of the methods described herein.

[0314] Tumor-associated peptide loading In some embodiments, the methods described herein further include contacting the APC with a plurality of tumor-associated peptides (e.g., neo-antigenic peptides). In some embodiments, the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) comprises more than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50 tumor-associated peptides (e.g., neo-antigenic peptides). In some embodiments, the APC is contacted with the composition comprising the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) for about 4 to about 24 hours.

[0315] In some embodiments, the APCs are pre-incubated with a composition comprising multiple tumor-associated peptides (e.g., neo-antigenic peptides) prior to use in the methods of activating immune cells described herein.

[0316] An exemplary embodiment of contacting a population of APCs with multiple tumor-associated peptides (e.g., neo-antigenic peptides) includes pulsing a population of APCs with multiple tumor-associated peptides (e.g., neo-antigenic peptides). As known in the art, pulsing refers to a process in which cells, such as APCs, are mixed with a solution containing tumor-associated peptides (e.g., neo-antigenic peptides) and, optionally, subsequently removing the tumor-associated peptides (e.g., neo-antigenic peptides) from the mixture. The population of APCs can be contacted with multiple tumor-associated peptides (e.g., neo-antigenic peptides) for a few seconds, minutes, or hours, such as about 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, or more. The concentration of each neo-antigenic peptide used in the contacting step can be about 0.1, 0.5, 1, 2, 3, 5, or 10 μg / mL. In some embodiments, the concentration of the tumor-associated peptide (e.g., neo-antigenic peptide) is about 0.1-200 μg / mL, including, for example, about 0.1-0.5, 0.5-1, 1-10, 10-50, 50-100, 100-150, or 150-200 μg / mL.

[0317] In some embodiments, the population of APCs is contacted with a plurality of tumor-associated peptides (e.g., neo-antigenic peptides) in the presence of a composition that promotes uptake of the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) by APCs. In some embodiments, a compound, material, or composition may be included in the solution of the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) to promote peptide uptake by APCs. Compounds, materials, or compositions that promote uptake of the plurality of tumor-associated peptides (e.g., neo-antigenic peptides) by APCs include, but are not limited to, lipid molecules and peptides having a plurality of positively charged amino acids. In some embodiments, more than about 50%, 60%, 70%, 80%, 90%, or 95% of the tumor-associated peptides (e.g., neo-antigenic peptides) are taken up by the population of APCs. In some embodiments, more than about 50%, 60%, 70%, 80%, 90%, or 95% of the APCs in the population take up at least one tumor antigen peptide.

[0318] immune cells The immune cells described herein can be any type of immune cell that can interact with APCs, be activated by APCs, and then exert their desired functions. Exemplary immune cells include T cells.

[0319] T cells or T lymphocytes play a central role in cell-mediated immunity. Each clone of activated T cells expresses a distinct T cell receptor (TCR) on its surface that is responsible for the recognition of antigens bound to MHC molecules on APCs and target cells (such as cancer cells). T cells are subdivided into several types, each expressing a unique combination of surface proteins and each having a distinct function.

[0320] Cytotoxic T cells (TCs) are involved in the immune response against and destruction of tumor cells and other infected cells, such as virus-infected cells. In general, TC cells function by recognizing class I MHC-presented antigens on APCs or any target cells. Stimulation of the TCR, together with co-stimulatory factors (e.g., CD28 on T cells binding to B7 on APCs, or stimulation by helper T cells), leads to activation of the TC cells. The activated TC cells can then proliferate and release cytotoxins, thereby destroying APCs or target cells (such as cancer cells). Mature TC cells generally express the surface proteins CD3 and CD8. Cytotoxic T cells belong to CD3+CD8+ T cells.

[0321] Helper T cells (TH) are T cells that help the activity of other immune cells by releasing T cell cytokines, which can regulate or suppress immune responses, induce cytotoxic T cells, and maximize the cell killing activity of macrophages. In general, TH cells function by recognizing class II MHC-presented antigens on APCs. Mature TH cells express the surface proteins CD3 and CD4. Helper T cells belong to CD3+CD4+ T cells.

[0322] Regulatory T cells (T REG T cells (T cells) generally regulate the immune system by promoting tolerance to self-antigens, thereby limiting autoimmune activity. In cancer immunotherapy, T REG contributes to the escape of cancer cells from the immune response. REG The cells generally express CD3, CD4, CD7, CD25, CTLA4, GITR, GARP, FOXP3 and / or LAP. CD4+CD25+Foxp3+ T cells are T REG It is a class of cells.

[0323] Memory T cells (Tm) are T cells that have previously encountered and responded to their specific antigen, or that have differentiated from activated T cells. Tumor-specific Tm constitute a small fraction of the total T cell mass, but play a critical function in the surveillance of tumor cells throughout the human lifespan. When tumor-specific Tm encounter tumor cells expressing their specific tumor antigen, the Tm are immediately activated and clonally expanded. The activated and expanded T cells differentiate into effector T cells and kill tumor cells with high efficiency. Memory T cells are important for establishing and maintaining long-term tumor antigen-specific responses of T cells.

[0324] Typically, antigens for T cells are protein molecules or linear fragments of protein molecules that can be recognized by the T cell receptor (TCR) to elicit a specific T cell response. Antigens can be derived from foreign sources, such as virus-encoded proteins, or endogenous sources, such as cell surface-expressed proteins. The smallest fragment of an antigen that is directly involved in the interaction with a specific TCR is known as an epitope. Multiple epitopes can be present in a single antigen, with each epitope being recognized by a separate TCR encoded by a particular clone of T cells.

[0325] To be recognized by TCR, antigen peptides or antigen fragments are processed into epitopes by APCs (such as dendritic cells) and then bind in an extended conformation within major histocompatibility complex (MHC) molecules to form MHC-peptide complexes on the surface of APCs (such as dendritic cells). MHC molecules are also known as human leukocyte antigens (HLA). MHC provides an extended binding surface for strong association between TCR and epitopes, while the combination of unique amino acid residues within the epitopes ensures the specificity of the interaction between TCR and epitopes. Human MHC molecules are classified into two types, MHC class I and MHC class II, based on their structural features, especially the length of the epitopes bound inside the corresponding MHC complex. MHC-I epitopes are epitopes that bind to and are presented by MHC class I molecules. MHC-II epitopes are epitopes that bind to and are presented by MHC class II molecules. MHC-I epitopes are typically about 8 to about 11 amino acids long, while MHC-II epitopes are about 13 to about 17 amino acids long. Due to genetic polymorphisms, various subtypes of both MHC class I and MHC class II molecules exist in the human population. T cell responses to specific antigen peptides presented by MHC class I or MHC class II molecules on APCs are known as MHC-restricted T cell responses.

[0326] In some embodiments, the immune cells are selected from the group consisting of PBMCs, tumor infiltrating T cells (TILs), and T cells (eg, CD4 T cells and / or CD8 T cells).

[0327] In some embodiments, the immune cells are PBMCs.

[0328] In some embodiments, the immune cells are tumor-infiltrating T cells (TILs).

[0329] In some embodiments, the immune cells are CD4 T cells and / or CD8 T cells.

[0330] In some embodiments, the immune cells and APCs are from the same individual. In some embodiments, the immune cells and APCs are from different individuals.

[0331] Preparation of activated immune cells (e.g. activated T cells) The methods described herein include co-culturing a population of immune cells (e.g., T cells) with a population of APCs described herein that are loaded with a plurality of tumor-associated peptides (e.g., neo-antigenic peptides).

[0332] In some embodiments, the co-culture is carried out for at least 24 hours. In some embodiments, the co-culture is carried out for at least about 1-5 days (e.g., about 1-3 days). In some embodiments, a population of immune cells (e.g., T cells) and a population of APCs loaded with a plurality of tumor-associated peptides (e.g., neo-antigen peptides) are co-cultured for any of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In some embodiments, a population of immune cells (e.g., T cells) are co-cultured with a population of APCs loaded with a plurality of tumor-associated peptides (e.g., neo-antigen peptides) for about 14 to about 21 days. In some embodiments, a population of immune cells (e.g., T cells) are co-cultured with a population of APCs loaded with a plurality of tumor-associated peptides (e.g., neo-antigen peptides) for about 14 days.

[0333] The population of immune cells (e.g., T cells) used in any embodiment of the methods described herein may be derived from various sources. A convenient source of immune cells is derived from PBMCs of human peripheral blood. For example, a population of T cells can be isolated from PBMCs, or alternatively, a population of PBMCs enriched for T cells (such as by addition of T cell-specific antibodies and cytokines) can be used in co-culture. In some embodiments, the population of T cells used in co-culture is obtained from the non-adherent fraction of peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are obtained by density gradient centrifugation of a sample of peripheral blood. In some embodiments, the population of activated T cells is prepared by obtaining a population of non-adherent PBMCs and co-culturing the population of non-adherent PBMCs with a population of APCs loaded with multiple tumor-associated peptides (e.g., neo-antigen peptides) (e.g., in the presence of at least one cytokine (such as IL-2) and anti-CD3 antibodies).

[0334] The co-culture may further include cytokines and other compounds to promote activation, maturation and / or proliferation of T cells, as well as to prime the T cells for subsequent differentiation, e.g., into memory T cells. Exemplary cytokines that may be used in this step include, but are not limited to, IL-7, IL-15, IL-21, and the like. Certain cytokines may promote T cell proliferation in the population of activated T cells in the co-culture. REG For example, in some embodiments, a high dose (e.g., about any of 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, or 1500 U / ml) of a cytokine (such as IL-2) can be used to co-culture a population of T cells with a population of dendritic cells loaded with multiple tumor antigen peptides to inhibit a low percentage of T REG Obtain a population of activated T cells with the cells.

[0335] In some embodiments, the method of activating immune cells includes two or more rounds of co-culture (e.g., two, three or four rounds) of immune cells (e.g., T cells) with the APC population. In some embodiments, each round takes about 6-8 days. In some embodiments, the first, second, third and / or fourth rounds do not include the addition of anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the immune cells (e.g., T cells) exhibit non-exhaustive characteristics after two, three or four rounds of co-culture. In some embodiments, the immune cells (e.g., T cells) can expand about 50-100-fold (e.g., at least 50-fold) after each round of culture. In some embodiments, each round takes about 5-10 days or 6-8 days. In some embodiments, the number of immune cells (e.g., T cells) after three or four rounds of co-culture is about 10 10 reaches.

[0336] In some embodiments, the methods of activating immune cells described herein further comprise expanding the population of immune cells after the co-culturing step. In some embodiments, expanding the population of immune cells comprises contacting the immune cells with a cytokine selected from the group consisting of IL-2, IL-7 and IL-15, optionally for about 2 days to about 10 days. In some embodiments, the co-culturing is performed in the presence of an anti-CD3 antibody and multiple cytokines, such as IL-2, IL-7, IL-15, IL-21, or any combination thereof.

[0337] The present application also provides populations of activated immune cells obtained by the methods described in this section.

[0338] V. Treatment Method The present application also provides a method of treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient, comprising administering to the patient a population of APCs and / or activated immune cells obtained by the above method.

[0339] In some embodiments, methods are provided for treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient comprising administering to the patient a population of APCs (e.g., any of those described in Section III or produced according to the methods described in Section II).

[0340] In some embodiments, a method of treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient is provided, comprising administering to the patient a population of antigen presenting cells (APCs), wherein the APCs are derived from monocytes obtained from an individual (e.g., a cancer patient or a viral infection patient), and wherein the APCs a) express high levels of one or more antigen presenting molecules, wherein the antigen presenting molecules are selected from the group consisting of MHCl, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, and / or b) low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2), and Siglec 10. In some embodiments, the monocytes exhibit lower expression levels of M-CSFR, GM-CSFR, IL-6R, IL-10R and / or IL-4R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from the individual compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the method further comprises administering a second therapy (e.g., radiation therapy) that induced immunogenic cell death. In some embodiments, the method comprises concurrently or subsequently administering the APCs and administering radiation therapy. In some embodiments, the APCs are not preloaded with disease- or condition-associated antigens (e.g., tumor antigens or viral antigens) prior to administration. In some embodiments, the APCs are preloaded with disease- or condition-associated antigens (e.g., tumor antigens or viral antigens) prior to administration.

[0341] In some embodiments, a method of treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient is provided, comprising administering to the patient a population of antigen presenting cells (APCs), the APCs being derived from monocytes obtained from an individual (e.g., a cancer patient or a viral infection patient), the APCs being obtained by a) separately or simultaneously contacting a population of monocytes obtained from the individual with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs. In some embodiments, the method further comprises administering a second therapy (e.g., radiation therapy) that induced immunogenic cell death. In some embodiments, the method comprises administering APCs and administering radiation therapy simultaneously or subsequently. In some embodiments, the APCs are not preloaded with disease or condition-related antigens (e.g., tumor antigens or viral antigens) before administration. In some embodiments, the APCs are preloaded with disease or condition-related antigens (e.g., tumor antigens or viral antigens) before administration.

[0342] In some embodiments, a method of treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient is provided, comprising administering to the patient a population of antigen presenting cells (APCs), wherein the APCs are derived from monocytes obtained from an individual (e.g., a cancer patient or a viral infection patient), and wherein the APCs a) express high levels of one or more antigen presenting molecules, wherein the antigen presenting molecules are selected from the group consisting of MHCl, MHCII, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, and / or b) low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from the group consisting of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2), and Siglec 10, which have been preloaded with a disease or condition-associated antigen (e.g., a tumor antigen or a viral antigen) prior to administration. In some embodiments, the monocytes exhibit lower expression levels of M-CSFR, GM-CSFR, IL-6R, IL-10R and / or IL-4R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) when obtained from an individual compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0343] In some embodiments, a method of treating a disease or condition (e.g., cancer, e.g., a viral infection) in a patient is provided, comprising administering to the patient a population of activated immune cells, wherein the immune cells are subjected to co-culture with a population of APCs, wherein the APCs are generated following contact with an IL-10 receptor activator (IL-10R activator) and one or more of an IFNgamma receptor activator (IFNR activator), a TNFalpha receptor activator (TNFR activator) and an IL-4 receptor activator (IL-4R activator), and wherein the APCs are pre-loaded with one or more peptides associated with the disease or condition (e.g., tumor associated peptides, e.g., neo-antigenic peptides, virus-specific peptides) prior to co-culture.

[0344] In some embodiments, a method of treating a disease or condition (e.g., cancer, e.g., viral infection) in a patient is provided, comprising administering to the patient a population of activated immune cells (e.g., T cells), the immune cells being subjected to co-culture with a population of APCs, the APCs being produced after contact with IL-10 and one or more of IFNγ, TNFα and IL-4, and the APCs being pre-loaded with one or more peptides (e.g., tumor-associated peptides, e.g., neo-antigenic peptides, virus-specific peptides) associated with the disease or condition prior to co-culture. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are T cells (e.g., CD3 T cells, e.g., CD4 T cells, e.g., CD8 T cells, e.g., both CD4 T cells and CD8 T cells, e.g., TILs) obtained from the patient's peripheral blood. In some embodiments, the immune cells are T cells (e.g., CD3 T cells, e.g., CD4 T cells, e.g., CD8 T cells, e.g., both CD4 and CD8 T cells, e.g., TILs) obtained from peripheral blood of an individual different from the patient (optionally with matching HLA types). In some embodiments, the APCs and activated immune cells are from the same individual. In some embodiments, the APCs and activated immune cells are from different individuals (optionally with matching HLA types). In some embodiments, the APCs are generated after contact with IL-10, IFNγ, TNFα, and IL-4. In some embodiments, the APCs are generated after contact with IL-10, IFNγ, TNFα, GM-CSF, IL-6, and IL-4. In some embodiments, the APCs are generated after contact with one or more refining factors described in Section II. In some embodiments, the activated immune cells are administered intratumorally, intraperitoneally, or intravenously. In some embodiments, the activated immune cells are administered at about 10 per dose. 7 ~10 9In some embodiments, the treatment methods described herein further include treating the patient with chemotherapy, radiation therapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with irradiation. In some embodiments, the site of irradiation is different from the site of the cancer being treated.

[0345] In some embodiments, a method of treating a virus-associated cancer in a patient is provided, comprising administering a population of activated T cells to the patient, the T cells being subjected to co-culture with a population of APCs, the APCs being produced after contact with IL-10 and one or more of IFNγ, TNFα and IL-4, the APCs being pre-loaded with one or more tumor-associated peptides (e.g., neo-antigenic peptides) associated with the virus-associated cancer prior to co-culture, and the virus antigen-reactive T cells being removed from the activated T cell population prior to administration. In some embodiments, the APCs are derived from the patient. In some embodiments, the activated T cells are derived from the patient. In some embodiments, the APCs and the activated T cells are both derived from the patient. In some embodiments, the activated immune cells are administered intratumorally, intraperitoneally or intravenously. In some embodiments, the activated immune cells are administered at a concentration of about 10 per dose. 7 ~10 9 In some embodiments, the treatment methods described herein further include treating the patient with chemotherapy, radiation therapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with irradiation. In some embodiments, the site of irradiation is different from the site of the cancer being treated.

[0346] In some embodiments, a method of treating a liver cancer associated with a virus (e.g., Hepatitis B virus or Hepatitis C virus) in a patient is provided, comprising administering a population of activated T cells to the patient, the T cells being subjected to co-culture with a population of APCs, the APCs being produced after contact with IL-10 and one or more of IFNγ, TNFα and IL-4, the APCs being pre-loaded with one or more tumor-associated peptides (e.g., neo-antigen peptides) prior to co-culture, and the viral antigen-reactive T cells being removed from the activated T cell population prior to administration. In some embodiments, the APCs are derived from the patient. In some embodiments, the activated T cells are derived from the patient. In some embodiments, the APCs and the activated T cells are both derived from the patient. In some embodiments, the activated immune cells are administered intratumorally, intraperitoneally or intravenously. In some embodiments, the activated immune cells are administered at a concentration of about 10 per dose. 7 ~10 9 In some embodiments, the treatment methods described herein further include treating the patient with chemotherapy, radiation therapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with irradiation. In some embodiments, the site of irradiation is different from the site of the cancer being treated.

[0347] patient In some embodiments, the patient has a solid tumor, hi some embodiments, the patient has a hematological cancer.

[0348] In some embodiments, the patient has advanced cancer. In some embodiments, the patient has late stage cancer. In some embodiments, the patient has stage II, III or IV cancer. In some embodiments, the patient has inoperable tumors and / or metastases. In some embodiments, the patient is a terminal patient.

[0349] In some embodiments, the patient is female. In some embodiments, the patient is male.

[0350] In some embodiments, the patient is a human. In some embodiments, the patient is at least about 50, 55, 60, 65, 70, or 75 years of age.

[0351] cancer As discussed above, treatment methods involving immune cells (e.g., T cells) activated by APCs produced by the various methods described herein are applicable to all types of cancer.

[0352] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0353] In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a late stage cancer. In some embodiments, the cancer is in stage II, III or IV. In some embodiments, the cancer is an inoperable tumor and / or is malignant.

[0354] In some embodiments, the cancer has undergone and / or failed one or more prior therapies (e.g., immune checkpoint blockade therapy (e.g., PD-1 antibodies), chemotherapy, surgery, cellular therapy (e.g., allogeneic NK cell infusion therapy)).

[0355] In some embodiments, the cancer is a recurrent or refractory cancer.

[0356] Examples of cancers described herein include adrenocortical carcinoma, primary myelofibrosis, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway tumor, and hypothalamic glioma, glioblastoma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary site, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic neoplasia, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatocellular carcinoma) and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g. small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), lymphatic tumors (e.g. lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndromes, myelodysplastic syndromes, myelodysplastic / myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine carcinoma, oropharyngeal cancer, ovarian cancer (e.g. ovarian epithelial carcinoma, ovarian germ cell tumors, ovarian low malignant tumors, etc.), large-scale tumors), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g. non-melanoma (e.g. squamous cell carcinoma), melanoma, Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor,These include, but are not limited to, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0357] In some embodiments, the cancer is a viral infection-associated cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-associated cancer (e.g., HPV-associated cervical cancer, e.g., HPV-associated head and neck cancer, e.g., HPV-associated squamous cell carcinoma). In some embodiments, the cancer is a human herpesvirus 8 (HHV8)-associated cancer (e.g., Kaposi's sarcoma). In some embodiments, the cancer is a human T-lymphoproliferative virus (HTLV-1)-associated cancer (e.g., adult T-cell leukemia or lymphoma). In some embodiments, the cancer is an Epstein-Barr virus (EBV)-associated cancer (e.g., Burkitt's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma, gastric cancer). In some embodiments, the cancer is a hepatitis B virus (HBV)-associated cancer (e.g., liver cancer). In some embodiments, the cancer is a hepatitis C virus)-associated cancer (e.g., liver cancer, non-Hodgkin's lymphoma).

[0358] Dosage and administration methods for activated immune cells (e.g., T cells) Activated immune cells can be administered in any desired dosage, including in some aspects a desired dose or number of cells or cell types. Thus, in some embodiments, the dosage of cells is based on the total number of cells (or number per kg of body weight) and / or the desired ratio of individual populations. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg of body weight) in individual populations or individual cell types.

[0359] In certain embodiments, activated immune cells (e.g., T cells, e.g., CD4 and / or CD8 T cells, e.g., TILs) are present in a range and / or amount of cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 10 million cells, about 20 million cells, about 30 million cells, about 40 million cells, about 5 ... cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million, about 250 million, about 350 million, about 450 million, about 650 million, about 800 million, about 900 million, about 3 billion, about 30 billion, about 45 billion), or any value between these ranges and / or per kilogram of body weight administered to the subject. Dosages may vary depending on the disease or disorder and / or attributes specific to the patient and / or other treatments.

[0360] In some embodiments, for example, when the subject is a human, the dose is about 1×10 9 Fewer than 10 total activated immune cells, e.g., about 1 x 10 6 pieces~5×10 8 A range of such cells, e.g., 2 x 10 6 pieces, 5×10 6 pieces, 1×10 7 pieces, 5×10 7 pieces, 1×10 8 pieces, 5×10 8 pcs or 1×10 9 total such cells, or total activated immune cells ranging between any two of the aforementioned values.

[0361] In some embodiments, the activated immune cells are about 10 per dose. 7 ~10 9 The cells are administered in individual doses.

[0362] In some embodiments, the treatment method comprises administering a dose of about 1×10 6 ~1×10 9 (For example, 10 6 ~10 7 , 10 7 ~10 8 , or 10 8 ~10 9 ) total activated immune cells (e.g., total CD3 T cells, both CD4 T cells and CD8 T cells, CD4 T cells only, CD8 T cells only, or TILs).

[0363] In some embodiments, the dose of activated immune cells is administered to the subject as a single dose, or is administered only once within a period of two weeks, one month, three months, six months, one year or more.

[0364] In some embodiments, the dose of total activated immune cells is about 10 4 ~about 10 9 between about 10 cells / kilogram (kg) of body weight, e.g., about 10 5 ~10 6 cells / kg body weight, for example, about 1 x 10 5 cells / kg, approximately 1.5 x 10 5 Cells / kg, approximately 2 x 10 5 cells / kg, or approximately 1 x 10 6 For example, in some embodiments, the activated immune cells are about 10 4 ~about 10 9 between 10 T cells / kilogram (kg) of body weight, for example 10 5 ~10 6 between 1 x 10 T cells / kg body weight, for example 1 x 10 5 T cells / kg, 1.5 x 10 5 T cells / kg, 2 x 10 5 T cells / kg, or 1 x 10 6T cells / kg body weight, or within a certain margin of error therebetween.

[0365] In some embodiments, the activated immune cells are at or about 10 4 ~about 10 9 CD4 + and / or CD8 + Between cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 CD4 + and / or CD8 + Between cells / kg body weight, for example, 1 x 10 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 x 10 6 CD4 + and / or CD8 + cells / kg body weight, or within a certain margin of error therebetween.

[0366] In some embodiments, the activated immune cells are greater than and / or at least about 1×10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 CD4 + cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 10 6 CD8+ cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 , or about 9 × 106 In some embodiments, the activated immune cells are administered at about 10 T cells or within a certain margin of error therebetween. 8 ~about 10 12 Between 10 T cells or approximately 10 10 ~about 10 11 Between 10 T cells, approximately 8 ~about 10 12 Between 10 T cells or approximately 10 10 ~about 10 11 CD4 + Between cells and / or about 10 8 ~about 10 12 CD8 + Between cells or about 10 10 ~about 10 11 CD8 + The cells are administered at or within a certain margin of error between them.

[0367] For prevention or treatment of disease, the appropriate dosage may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the activated immune cells are administered for preventative or therapeutic purposes, previous treatments, the subject's clinical history and response to the activated immune cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0368] In some embodiments, the APCs described herein are from about 5000 to about 10,000 cells / mm of tumor mass. 3 is administered to the subject in the range of

[0369] In some embodiments, the size of the dose is determined based on one or more criteria, such as, for example, the subject's response to previous treatment (e.g., chemotherapy), the disease burden in the subject (e.g., tumor burden, volume, size, or extent, extent or type of metastasis, stage of disease, and / or the likelihood or incidence of the subject developing a toxic outcome (e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity), and / or the host immune response to the administered activated immune cells.

[0370] In some embodiments, the size of the dose is determined by the burden of disease or symptoms in the subject.For example, in some embodiments, the number of cells administered in a dose is determined based on the tumor burden present in the subject immediately before the administration of the first dose of cells.In some embodiments, the size of the first dose and / or subsequent doses is inversely correlated with disease burden.In some embodiments, such as in the context of high disease burden, the subject is administered a small number of cells.In other embodiments, such as in the context of lower disease burden, the subject is administered a larger number of cells.

[0371] The activated immune cells can be administered by any suitable means, for example, by bolus injection, injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, subtenon injection, retrobulbar injection, periocular injection, or posterior parascleral delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and, if desired for localized treatment, by intralesional administration. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the activated immune cells. In some embodiments, the activated immune cells are administered by multiple bolus administration, for example, over a period of 3 days or less, or by continuous infusion administration of the activated immune cells.

[0372] In some embodiments, the activated immune cells are administered intratumorally, intraperitoneally, or intravenously.

[0373] Combination therapy In some embodiments, the APCs (e.g., antigen-triggered or naive) or activated immune cells are administered as part of a combination treatment, concurrently, simultaneously, or sequentially with another therapeutic intervention (i.e., a second treatment), such as an antibody or engineered cell or receptor or agent, e.g., a cytotoxic agent or therapeutic agent. In some embodiments, the APCs or activated immune cells are administered prior to another therapeutic intervention. In some embodiments, the APCs or activated immune cells are administered after another therapeutic intervention. In some embodiments, the APCs or activated immune cells are co-administered with one or more additional therapeutic agents, or co-administered simultaneously, concurrently, or sequentially in any order in relation to another therapeutic intervention. In some situations, the APCs or activated immune cells are co-administered with another treatment close enough in time that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the APCs or activated immune cells are administered prior to one or more additional therapeutic agents. In some embodiments, the APCs or activated immune cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents to be administered include, for example, a cytokine (such as IL-2) to increase the persistence of activated immune cells. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0374] In some embodiments, the second therapy comprises chemotherapy, radiation therapy, or an immune checkpoint inhibitor. In some embodiments, the second therapy is a gene therapy (e.g., an mRNA-based gene therapy). In some embodiments, the second therapy comprises administration of a cancer vaccine (such as an mRNA-based cancer vaccine or a DNA-based cancer vaccine). In some embodiments, the second therapy comprises administration of an oncolytic virus. In some embodiments, the APCs or immune cells are administered prior to administration of the second therapy. In some embodiments, the APCs or immune cells are administered in a neoadjuvant environment.

[0375] In some embodiments, the second therapy comprises treating the patient with radiation, hi some embodiments, the site of radiation is different from the site of the cancer being treated.

[0376] Thus, for example, in some embodiments, a method of treating an individual having cancer is provided that comprises administering to the individual an effective amount of activated APCs or immune cells by any of the methods described herein, wherein the individual is treated with radiation therapy, the site of irradiation being different from the site of the cancer being treated.

[0377] In some embodiments, the radiation therapy is selected from the group consisting of external radiation therapy, internal radiation therapy (brachytherapy), intraoperative radiation therapy (IORT), total body radiation therapy, radioimmunotherapy, and administration of radiosensitizers and radioprotectors. In some embodiments, the radiation therapy is external beam radiation therapy, including three-dimensional conformal radiation therapy (3D-RT), intensity-modulated radiation therapy (IMRT), photon beam therapy, image-guided radiation therapy (IGRT), and stereotactic radiation therapy (SRT), as appropriate. In some embodiments, the radiation therapy is brachytherapy, including interstitial brachytherapy, intracavitary brachytherapy, intracavitary radiation therapy, and intravenously administered radioactive tagged molecules, as appropriate.

[0378] VI. Compositions Comprising Multiple Survival, Differentiation and / or Maturation Factors ("S / D / M Factors") The present application also provides a composition (e.g., a cell culture medium) comprising a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the plurality of S / D / M factors is configured as described above.

[0379] In some embodiments, a composition (e.g., cell culture medium) is provided that includes multiple survival, differentiation and / or maturation factors ("S / D / M factors"): 1) an IL-10 receptor (IL-10R) activator, and 2) one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator.

[0380] In some embodiments, the IL-10R activator is selected from the group consisting of IL-10 (e.g., pegylated IL-10, e.g., pegylodecacin or AM0010), IL-10 family members (e.g., IL-19, IL-20, IL-22, IL-24, IL-26, IL-28), IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of IL-10R downstream of STAT3 (e.g., long non-coding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). In some embodiments, the IL-10R activator is IL-10. In some embodiments, the IL-10 is human IL-10 or human recombinant IL-10. In some embodiments, IL-10 is present in the medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally from about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml).

[0381] In some embodiments, the IFNGR activator is selected from the group consisting of IFNγ, IFNGR agonist antibodies, and small molecule activators of IFNGR. In some embodiments, the IFNGR activator is IFNγ. In some embodiments, the IFNγ is human IFNγ or human recombinant IFNγ. In some embodiments, the IFNγ is present in the medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally at about 10 ng / ml to about 200 ng / ml (e.g., about 50 to 100 ng / ml).

[0382] In some embodiments, the IL-4R activator is selected from the group consisting of IL-4, IL-13, IL-4R agonist antibodies, and small molecule activators of IL-4R. In some embodiments, the IL-4R activator is IL-4. In some embodiments, the IL-4 is human IL-4 or human recombinant IL-4. In some embodiments, the IL-4 is present in the medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, and further optionally from about 30 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 1 ng / ml).

[0383] In some embodiments, the TNFR activator is selected from the group consisting of TNFα, TNFR agonist antibodies, and small molecule activators of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the TNFα is human TNFα or human recombinant TNFα. In some embodiments, the TNFα is present in the medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, and further optionally from about 0.5 ng / ml to about 30 ng / ml (e.g., from about 1 to 10 ng / ml).

[0384] In some embodiments, the plurality of S / D / M factors comprises two or more agents selected from the group consisting of an IL-4R activator, a TNFR activator, and an IFNGR activator. In some embodiments, the plurality of S / D / M factors comprises a TNFR activator and an IFNGR activator.

[0385] In some embodiments, the multiple S / D / M factors include IL-10, IL-4, TNFα, and IFNγ.

[0386] In some embodiments, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

[0387] In some embodiments, the GM-CSFR activator is selected from the group consisting of GM-CSF, GM-CSFR agonist antibodies, and small molecule activators of GM-CSFR. In some embodiments, the GM-CSFR activator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF or human recombinant GM-CSF. In some embodiments, the GM-CSF is present in the medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, and further optionally from about 100 pg / ml to about 1 ng / ml (e.g., from about 100 pg / ml to about 500 pg / ml, e.g., about 300 pg / ml).

[0388] In some embodiments, the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R. In some embodiments, the IL-6R activator is IL-6. In some embodiments, the IL-6 is human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, and further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10 to 50 pg / ml, e.g., about 30 pg / ml).

[0389] In some embodiments, the multiple S / D / M factors include IL-10, IL-4, TNFα, IL-6, GM-CSF and IFNγ.

[0390] In some embodiments, the multiple maturation factors further comprise one or more of IL-2, IL-4, IL-17, and M-CSF, agonist antibodies thereof, or small molecule activators thereof.

[0391] In some embodiments, a composition (e.g., cell culture medium) is provided that comprises an IL-10 receptor (IL-10R) activator, optionally where the IL-10R activator is IL-10 (e.g., human IL-10, or human recombinant IL-10), and further optionally where the IL-10 is present in the medium at a concentration of at least about 2 ng / ml (e.g., at least about 10 ng / ml, e.g., at least about 20 ng / ml, e.g., from about 10 ng / ml to about 200 ng / ml, e.g., about 20 ng / ml), and optionally where the medium is specifically for cancer cells (e.g., monocytes obtained from a cancer patient) or cells (e.g., monocytes) that express low levels of IL-10R (e.g., at least 20%, 30%, 40%, 50% lower than the corresponding cells of a reference individual (e.g., a healthy individual)).

[0392] The compositions described herein may be prepared by combining each component into a single composition. In some embodiments, the compositions are prepared by culturing immune cells (e.g., T cells, e.g., CD4 T cells, e.g., CD8 T cells) and obtaining cell culture supernatant. In some embodiments, the supernatant can be further supplemented with additional components (or additional amounts of components already present in the supernatant) or modified to remove components to obtain the desired composition.

[0393] In some embodiments, a composition (e.g., cell culture medium) is provided that is derived from a culture (e.g., supernatant) of T cells after treatment with anti-CD3 and anti-CD28 antibodies, where the medium comprises IL-10. In some embodiments, the T cells are CD4 T cells. In some embodiments, the T cells are CD8 T cells. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the T cells are isolated from PBMCs of the same or a different individual and have been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment. In some embodiments, the medium is derived from a culture of T cells after treatment with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally for about 2 days. In some embodiments, at least one or more molecules (e.g., IL-2) are removed from the culture of T cells. In some embodiments, the one or more molecules are selected from the group consisting of IL-2, M-CSF, IL-12, and IL-17 (eg, IL-17A).

[0394] Exemplary embodiments Embodiment 1. A method of stimulating a population of monocytes from an individual to produce a population of antigen presenting cells ("APCs"), comprising separately or simultaneously contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising one or more agents selected from the group consisting of 1) an IL-10 receptor (IL-10R) activator, and 2) an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator and an interferon gamma (IFNγ) receptor (IFNGR) activator, thereby obtaining a population of APCs.

[0395] Embodiment 2. The method of embodiment 1, wherein the IL-10R activator is selected from the group consisting of IL-10, an IL-10 family member, an IL-10R agonist antibody, a small molecule activator of IL-10R and an activator of downstream STAT3 of IL-10R, optionally wherein the activator of downstream STAT3 of IL-10R is selected from an IL-10 family cytokine, an IL-12 family cytokine, an IL-6 family cytokine, a small molecule STAT3 activator and G-CSF.

[0396] Embodiment 3. The method of embodiment 1, wherein the IL-10R activator is selected from the group consisting of IL-10, IL-22, IL-19, IL20, IL-24, IL12, IL-23, IL-6, colivelin TFA, garcinone D and G-CSF, optionally wherein the IL-10R activator is IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, colivelin TFA or garcinone D.

[0397] Embodiment 4. The method of any one of embodiments 1-3, wherein the plurality of S / D / M factors comprises an IL-4R activator, and optionally the IL-4R activator is selected from the group consisting of IL-4, an IL-4R agonist antibody, and a small molecule activator of IL-4R.

[0398] Embodiment 5. The method of embodiment 4, wherein the IL-4R activator is IL-4.

[0399] Embodiment 6. The method of any one of embodiments 1-5, wherein the plurality of S / D / M factors comprises a TNFR activator, and optionally the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR.

[0400] Embodiment 7 The method of embodiment 6, wherein the TNFR activator is TNFα.

[0401] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the plurality of S / D / M factors comprises an IFNGR activator, and optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.

[0402] Embodiment 9 The method of embodiment 8, wherein the IFNGR activator is IFNγ.

[0403] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the multiple S / D / M factors are present in a single composition.

[0404] Embodiment 11. The method of any one of embodiments 1-10, wherein at least one of the plurality of S / D / M factors is provided separately from at least one other S / D / M factor of the plurality of S / D / M factors.

[0405] Embodiment 12. The method of any one of embodiments 1-11, wherein the multiple S / D / M factors comprise two or more agents selected from the group consisting of IL-4R activators, TNFR activators and IFNGR activators.

[0406] Embodiment 13. The method of embodiment 12, wherein the plurality of S / D / M factors comprises an IL-10R activator, TNFα and IFNγ, optionally wherein the plurality of S / D / M factors comprises an IL-10 family cytokine (e.g., IL-10, IL-22, IL-19, IL-24, IL-20, IL-26), TNFα and IFNγ, optionally wherein the plurality of S / D / M factors comprises an IL-10R activator, IL-4, TNFα and IFNγ.

[0407] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

[0408] Embodiment 15. The method of embodiment 14, wherein the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR.

[0409] Embodiment 16 The method of embodiment 15, wherein the GM-CSFR activator is GM-CSF.

[0410] Embodiment 17. The method of any one of embodiments 1-16, wherein the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R.

[0411] Embodiment 18. The method of embodiment 17, wherein the IL-6R activator is IL-6.

[0412] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the plurality of S / D / M factors is derived from a culture of T cells after treatment with an anti-CD3 antibody and an anti-CD28 antibody, and optionally the plurality of S / D / M factors is derived from a supernatant of the culture.

[0413] Embodiment 20. The method of embodiment 19, wherein the T cells are isolated from PBMCs of the same or a different individual, and optionally the T cells have not been previously treated with anti-CD3 and / or anti-CD28 antibodies prior to treatment.

[0414] Embodiment 21. The method of embodiment 19 or embodiment 20, wherein the plurality of S / D / M factors are derived from culture after the T cells have been treated with anti-CD3 and anti-CD28 antibodies for about 1-3 days, optionally about 2 days.

[0415] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the monocytes are cultured in the presence of S / D / M factors or medium derived from a culture of T cells for about 2 to 3 days.

[0416] Embodiment 23. The method of any one of embodiments 1 to 22, further comprising contacting the population of monocytes with a plurality of refining factors selected from the group consisting of type I interferon, IFNγ, TNFα, a TLR ligand, CD40L or CD40 ligation antibody, an anti-PD-L1 antibody and TPI-1, wherein optionally the type I interferon comprises IFNα and / or IFNβ, and optionally the TLR ligand is Poly IC, CpG or LPS.

[0417] Embodiment 24. The method of embodiment 23, wherein the plurality of refinement factors are provided after contacting the plurality of monocytes with the plurality of S / D / M factors or medium derived from a culture of T cells, thereby producing a population of APCs, and the population of APCs is cultured in the presence of the plurality of refinement factors for about 1-5 days, and optionally the population of APCs is cultured for about 1 day.

[0418] Embodiment 25. The method of embodiment 23 or embodiment 24, wherein the multiple refining factors are provided when: a) at least about 50% of the monocytes are viable; b) at least about 30% of the population of APCs exhibit dendritic cell morphology; and / or c) the population of APCs expresses i) high levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86 and / or CD40, and / or ii) low levels of SIRPα.

[0419] Embodiment 26 The method of any one of embodiments 23 to 25, wherein the refining factors include IFNα, IFNγ and TNFα.

[0420] Embodiment 27. The method of embodiment 26, wherein the refining agent further comprises at least two agents selected from the group consisting of PolyIC, CpG, CD40L, R848 and an anti-PD-L1 antibody, and optionally, the refining agent comprises a SHP-1 inhibitor (e.g., TPI-1).

[0421] Embodiment 28. A method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium having one or more molecules that promote IL-10 receptor (IL-10R) expression on monocytes.

[0422] The method of embodiment 29.1 or more molecules comprise an IL-10R activator, optionally wherein the IL-10R activator is selected from the group consisting of IL-10, an IL-10 family member, an IL-10R agonistic antibody, a small molecule activator of IL-10R, and an activator of IL-10R downstream of STAT3, and further optionally wherein the IL-10R activator is IL-10.

[0423] Embodiment 30. A method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium having an IL-10R activator, wherein the IL-10R activator may be selected from the group consisting of IL-10, an IL-10 family member, an IL-10R agonistic antibody, a small molecule activator of IL-10R, and an activator of IL-10R downstream of STAT3, and further optionally wherein the IL-10R activator is IL-10.

[0424] Embodiment 31 The method of any one of embodiments 28 to 30, wherein the population of monocytes expresses low levels of IL-10R prior to contact with the molecule.

[0425] Embodiment 32. The method of any one of embodiments 28-31, wherein the culture comprises a TNFα receptor (TNFR) activator and / or an interferon gamma (IFNγ) receptor (IFNGR) activator, optionally wherein the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, optionally wherein the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally wherein the culture comprises TNFα and / or IFNγ.

[0426] Embodiment 33. A method of increasing expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual with cancer, comprising contacting the population of monocytes with one or more agents selected from the group consisting of IL-10R activators, TNFR activators and IFNGR activators.

[0427] Embodiment 34. A method of promoting survival of a population of monocytes from an individual in in vitro culture, comprising culturing the population of monocytes in a medium comprising IL-10, TNFα and IFNγ.

[0428] Embodiment 35. A method of promoting differentiation of a population of monocytes into individual antigen presenting cells ("APCs") in in vitro culture comprising culturing the population of monocytes in a medium having one or more molecules selected from the group consisting of IL-4 receptor (IL-4R) activators, TNFα receptor (TNFR) activators and interferon gamma (IFNγ) receptor (IFNGR) activators.

[0429] Embodiment 36 The method of embodiment 35, wherein the culture further comprises an IL-6 receptor (IL-6R) activator and / or a GM-CSF receptor (GM-CSFR) activator.

[0430] Embodiment 37 The method of any one of embodiments 1 to 36, wherein a plurality of monocytes are obtained from the peripheral blood of the individual, and optionally the monocytes express CD14 and are obtained from the peripheral blood.

[0431] Embodiment 38 The method of any one of embodiments 1 to 37, wherein the individual has cancer.

[0432] Embodiment 39. The method of embodiment 38, wherein the individual has a late stage cancer.

[0433] Embodiment 40 The method of any one of embodiments 1 to 39, wherein the individual has a solid tumor.

[0434] Embodiment 41. The method of any one of embodiments 1 to 40, wherein the individual has an inoperable tumor and / or metastasis.

[0435] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the individual is a human.

[0436] Embodiment 43. A population of APCs produced by the method of any one of embodiments 1 to 27 and 35 to 42.

[0437] Embodiment 44. A population of APCs, the APCs being a) MHC-I+ / high, MHC-II+ / high and CD40+ / high, b) TLR2+ / high and / or STING+ / high, and c) LOX1+ / high and / or uPAR+ / high, and optionally wherein the expression level of CD40 on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages and / or MoDCs.

[0438] Embodiment 45. A population of APCs, wherein the APCs express higher levels of one or more antigen presenting molecules, the antigen presenting molecules being selected from the group consisting of MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL and CD40, than dendritic cells obtained from healthy humans and cultured with GM-CSF and IL-4 for about 5 days, optionally the APCs are generated from monocytes in ex vivo cell culture, further optionally the monocytes are obtained from a cancer patient, optionally the APCs express lower levels of an inhibitory signaling molecule, the inhibitory signaling molecule being selected from the group consisting of TGFβR, SIRPα, LIlRB and Siglec 10.

[0439] Embodiment 46. A method for activating a population of immune cells, comprising co-culturing the population of immune cells with a population of APCs according to any one of embodiments 43 to 45, wherein the APCs are pre-loaded with one or more neo-antigenic peptides.

[0440] Embodiment 47 The method of embodiment 46, comprising contacting the APCs with a composition comprising a plurality of neo-antigenic peptides and / or the APCs have been pre-incubated with the composition.

[0441] Embodiment 48. The method of embodiment 47, wherein the composition comprising a plurality of neo-antigenic peptides is a surgical resection of tumor tissue or a biopsy extract thereof.

[0442] Embodiment 49. The method of embodiment 47, wherein the composition comprising a plurality of neo-antigenic peptides is a mixture of tumor cells or extracts thereof isolated from tumor tissue or biopsy.

[0443] Embodiment 50. The method of embodiment 49, wherein the composition comprising a plurality of neo-antigenic peptides is a mixture of isolated neo-antigenic peptides.

[0444] Embodiment 51 The method of embodiment 50, wherein the isolated neo-antigenic peptide is a synthetic peptide.

[0445] Embodiment 52. The method of any one of embodiments 47-51, wherein the APCs are contacted with a composition comprising a plurality of neo-antigenic peptides for about 4 to about 24 hours.

[0446] Embodiment 53. The method of any one of embodiments 46-52, wherein the immune cells are selected from the group consisting of PBMCs, tumor infiltrating T cells (TILs) and T cells, and optionally the immune cells are T cells, and optionally the T cells are CD8 T cells and / or CD4 T cells, and optionally the activation is performed at least three times, each time of co-culture takes at least about 5 days, and at least two of the three co-cultures do not comprise an anti-CD3 antibody or an anti-CD28 antibody.

[0447] Embodiment 54. The method of any one of embodiments 46 to 53, wherein the co-culture is carried out for at least 24 hours.

[0448] Embodiment 55. The method of any one of embodiments 46 to 54, further comprising expanding the population of immune cells after the co-culturing step.

[0449] Embodiment 56. The method of embodiment 55, wherein expanding the population of immune cells comprises contacting the immune cells with a cytokine selected from the group consisting of IL-2, IL-7 and IL-15, optionally for about 2 to about 10 days.

[0450] Embodiment 57. The method of any one of embodiments 46 to 56, wherein the population of immune cells and the antigen-presenting cells are derived from the same individual.

[0451] Embodiment 58. The method of any one of embodiments 46 to 57, wherein the population of immune cells and the antigen-presenting cells are not derived from the same individual.

[0452] Embodiment 59. A population of activated immune cells obtained by the method of any one of embodiments 46 to 58.

[0453] Embodiment 60. A method for treating cancer in a patient, comprising administering to the patient a population of APCs of embodiments 43-45 and / or activated immune cells of embodiment 59.

[0454] Embodiment 61 The method of embodiment 60, wherein the APCs or activated immune cells are administered intratumorally, intraperitoneally or intravenously.

[0455] Embodiment 62. The activated immune cells are about 10 per dose. 7 ~10 9 62. The method of embodiment 61, wherein the cell is administered in individual cells.

[0456] Embodiment 63. The method of any one of embodiments 60-62, further comprising treating the patient with chemotherapy, radiation therapy, or an immune checkpoint inhibitor.

[0457] Embodiment 64. The method of embodiment 63, wherein the method comprises treating the patient with radiation.

[0458] Embodiment 65. The method of embodiment 64, wherein the irradiation site is different from the site of the cancer being treated.

[0459] Embodiment 66 The method of any one of embodiments 60 to 65, wherein the APCs or activated immune cells administered to the patient are derived from the patient.

[0460] Embodiment 67. The method of any one of embodiments 60 to 65, wherein the APCs or activated immune cells administered to the patient are not derived from the patient.

[0461] Embodiment 68. The method of any one of embodiments 60 to 67, wherein the cancer being treated is a solid tumor.

[0462] Embodiment 69. A composition comprising a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), the plurality of S / D / M factors comprising: 1) an IL-10 receptor (IL-10R) activator; and 2) one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon gamma (IFNγ) receptor (IFNGR) activator.

[0463] Embodiment 70. The composition of embodiment 69, wherein the IL-10R activator is selected from the group consisting of IL-10, an IL-10 family member, an IL-10R agonistic antibody, a small molecule activator of IL-10R and an activator of IL-10R downstream of STAT3.

[0464] Embodiment 71. The composition of embodiment 70, wherein the IL-10R activator is IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, colivelin TFA or garcinone D, optionally wherein the IL-10R activator is IL-10.

[0465] Embodiment 72. The composition of any one of embodiments 69 to 71, wherein the plurality of S / D / M factors comprises an IL-4R activator, and optionally the IL-4R activator is selected from the group consisting of IL-4, an IL-4R agonist antibody, and a small molecule activator of IL-4R.

[0466] Embodiment 73 The composition of embodiment 72, wherein the IL-4R activator is IL-4.

[0467] Embodiment 74. The composition of any one of embodiments 69 to 73, wherein the multiple S / D / M factors comprise a TNFR activator, and optionally the TNFR activator is selected from the group consisting of TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR.

[0468] Embodiment 75 The composition of embodiment 74, wherein the TNFR activator is TNFα.

[0469] Embodiment 76. A composition described in any one of embodiments 69 to 75, wherein the multiple S / D / M factors include an IFNGR activator, and optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.

[0470] Embodiment 77 The composition of embodiment 76, wherein the IFNGR activator is IFNγ.

[0471] Embodiment 78. The composition of any one of embodiments 69 to 77, wherein the multiple S / D / M factors comprise two or more agents selected from the group consisting of IL-4R activators, TNFR activators and IFNGR activators.

[0472] Embodiment 79. The composition of embodiment 78, wherein the multiple S / D / M factors include IL-10, IL-4, TNFα and IFNγ.

[0473] Embodiment 80. The composition of embodiments 69-79, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

[0474] Embodiment 81 The composition of embodiment 80, wherein the GM-CSFR activator is selected from the group consisting of GM-CSF, a GM-CSFR agonist antibody, and a small molecule activator of GM-CSFR.

[0475] Embodiment 82 The composition of embodiment 81, wherein the GM-CSFR activator is GM-CSF.

[0476] Embodiment 83. The composition of any one of embodiments 69 to 82, wherein the multiple S / D / M factors further comprise an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from the group consisting of IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R.

[0477] Embodiment 84. The composition of embodiment 83, wherein the IL-6R activator is IL-6. EXAMPLES

[0478] Working Example The following examples are purely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0479] Example 1. Experiments described herein studying peripheral monocytes from cancer patients with late-stage malignancies, e.g., stages II, III or IV with inoperable tumors and / or metastases, found that these monocytes (termed cancer monocytes or cMo) exhibited different responses to classical macrophage / DC differentiation factors M-CSF and / or GM-CSF compared to monocytes from healthy donors (healthy monocytes or Mo). For differentiation into macrophages (Figure 1A-1C), Mo are readily driven by M-CSF to differentiate into macrophages in 4-5 days, whereas cMo under the same treatment exhibited non-responsiveness (no cell adhesion or morphological changes) followed by cell death in 2 days. Similarly, cMo responded poorly to GM-CSF alone or GM-CSF+IL-4, which drives DC differentiation (data not shown). Over 20 cMo samples from cancer patients with a variety of solid tumors, including lung, renal, liver, colorectal, thymic, and sarcoma, have been tested, and in all cases, cMo consistently demonstrated unresponsiveness or hyporesponsiveness to M-CSF and GM-CSF, but with high rates of cell death (Figure 4). Parallel experiments testing the same M-CSF and GM-CSF reagents successfully differentiated Mo from healthy donors with viability rates of over 90%.

[0480] Further studies of M-CSF- and GM-CSF-mediated signaling as shown in Figure 2A confirmed that M-CSF and GM-CSF treatment induced strong survival signaling in Mo cells through activation of Akt and Erk1 / 2 (i.e., increased pAkt and pErk1 / 2 levels compared to total Akt and Erk1 / 2 protein levels, respectively). However, this signaling event was not replicated in cMo. Instead, in response to M-CSF and GM-CSF, cMo showed enhanced apoptotic signals and increased caspase-9 and caspase-3 cleavage, leading to cell death (Figure 2B). Examination of Akt and Erk1 / 2 as well as other signaling molecules detected similar total levels in Mo versus cMo (Figure 2C), ruling out a lack of protein phosphorylation due to reduced protein translation.

[0481] Examination of cell surface cytokine receptors revealed cMo compared to Mo expressing reduced levels of MCSF-R and GMCSF-R (Figure 1D), partially explaining the poor response of cMo to these differentiation factors by implying that insufficient stimulation intensity not only disables cell survival signaling but also induces apoptosis. The fact that cMo respond poorly to M-CSF and / or GM-CSF has also been reported by others (Gordon and Freedman, 2006; Ramos et al., 2012), and those results are consistent with the data presented herein. Previous transcriptional profiling analyses by different groups have also demonstrated various gene expression patterns between cMo (or called TEMo) and Mo from healthy donors (Bergenfelz et al., 2015; Cassetta et al., 2019; Chittezhath et al., 2014; Ramos et al., 2020).

[0482] Example 2. Although cMo could not be differentiated with M-CSF and GM-CSF, conditioned medium from TCR ligation-activated T cells was found to induce cMo differentiation into phagocytic APCs.

[0483] In these experiments (see Figure 3A), freshly isolated T cells from PBMCs from either healthy donors or autologous cancer patients were ligated with anti-CD3 and anti-CD28 antibodies to induce TCR activation in "autoimmune" CD4 T cells, which secreted abnormally high levels of IL-10 (>20 ng / ml) during the first round of anti-CD3 / anti-CD28 costimulation (Figure 3A-D). Two days later, T cell culture medium was used to treat cMo. In all experiments with cMo from various cancer patients (Figure 4), medium from TCR-activated T cells rapidly induced cMo adhesion to culture dishes (1-4 h). It was found that high levels of IL-10, together with other cytokines (e.g., IFNγ, TNFα, IL-4, GM-CSF, and IL-6) produced by the same CD4 T cells, could support the survival and differentiation of cMo into a unique type of APC (termed κ APC), as shown by changes in cell morphology (Figure 3E-G). Furthermore, this culture condition induced phenotypic differentiation into professional APC (Figure 3I). At 2 days, cMo-differentiated APCs showed increased expression of cell surface antigen presentation machinery, including high levels of MHC-I, MHC-II, costimulatory molecules CD80 and CD86, as well as APC activation molecules CD40 and PD-L1 (Figure 3E, high expression of MHC-II marked differentiation into professional APC).

[0484] Taken together, these results suggest that specific factors produced by TCR-stimulated T cells were able to both support cMo survival (adhesion) and differentiate cMo into professional APCs.TCR-stimulated T cell medium collected on day 2 was termed Karnelian X1 (or KX1).

[0485] Example 3. Further studies of Karnelian X1 found that biological components capable of supporting cMo survival and differentiation were present in fractions with molecular weights (MW) between 30KD and 100KD (fractions enriched in T cell-produced cytokines while excluding exosomes and lipid vesicles). Analysis of cytokines identified IFNγ, IL-2, IL-10, TNFα, GM-CSF, IL-6, IL-17, M-CSF and IL-4 (Figure 5A). Notably, high levels of IL-10 and IL-6 were detected in Karnelian X1. Further cytokine depletion assays confirmed the critical role of IL-10, whose depletion greatly (60-80%) reduced the capacity of cMo adhesion and survival (Figure 5B). Addition of IL-10 (recombinant) to IL-10-depleted Karnelian X1 restored its viability in a dose-dependent manner (Figure 5C). Inhibition of IL-10R-associated JAK with JAK inhibitor I, or downstream STAT3 with inhibitors C188-9 and napabucasin, dose-dependently abolished the effect of KX1 (Figure 5D), resulting in cMo apoptosis. In contrast, the addition of STAT3 activators colivelin, TFA, or garcinone D to IL-10-depleted KX1 medium (KX1 lowIL-10 ) restored cMo survival and promoted cMo differentiation into κAPCs (Figure 5E). Examination of other cytokines that activate STAT3 found that IL-10 family members, such as IL-19, IL-20, IL-22, and IL-24, as well as IL-12 family members (IL-12 and IL-23), could replace IL-10 to support cMo survival and differentiation (Figures 5F and 5G). The mechanism of IL-12 involves induction of IL-10 (Figure 5H). In addition, IL-12 is known to di...

Claims

1. A method for stimulating a population of individual-derived monocytes to produce a population of antigen-presenting cells ("APCs"), comprising the step of contacting the monocyte population separately or simultaneously with a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), Here, the plurality of S / D / M factors include 1) an IL-10 receptor (IL-10R) activator and 2) one or more drugs selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon-gamma (IFNγ) receptor (IFNGR) activator, and the individual has cancer. A method for obtaining a group of APCs.

2. The method according to claim 1, wherein the IL-10R activator is selected from the group consisting of IL-10, IL-10 family members, IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of downstream STAT3 of IL-10R, and optionally the activator of downstream STAT3 of IL-10R is selected from IL-10 family cytokines, IL-12 family cytokines, IL-6 family cytokines, small molecule STAT3 activators, and G-CSF.

3. The method according to claim 1, wherein the IL-10R activator is selected from the group consisting of IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, IL-6, Coliberin TFA, Garcinone D, and G-CSF, and optionally the IL-10R activator is IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, Coliberin TFA, or Garcinone D.

4. The method according to claim 1, wherein the plurality of S / D / M factors include an IL-4R activator, and optionally the IL-4R activator is selected from the group consisting of IL-4, an IL-4R agonist antibody, and a small molecule activator of IL-4R.

5. The method according to claim 4, wherein the IL-4R activator is IL-4.

6. The method according to claim 1, wherein the plurality of S / D / M factors include a TNFR activator, and optionally the TNFR activator is selected from the group consisting of TNFα, TNFR agonist antibodies, and small molecule activators of TNFR.

7. The method according to claim 6, wherein the TNFR activator is TNFα.

8. The method according to claim 1, wherein the plurality of S / D / M factors include an IFNGR activator, and optionally the IFNGR activator is selected from the group consisting of IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.

9. The method according to claim 8, wherein the IFNGR activator is IFNγ.

10. The method according to any one of claims 1 to 9, wherein the plurality of S / D / M factors are present in a single composition.

11. The method according to any one of claims 1 to 9, wherein the plurality of S / D / M factors comprises two or more agents selected from the group consisting of IL-4R activator, TNFR activator, and IFNGR activator.

12. The method according to claim 11, wherein the plurality of S / D / M factors include IL-10R activator, TNFα and IFNγ, and optionally the plurality of S / D / M factors include IL-10 family cytokines (e.g., IL-10, IL-22, IL-19, IL-24, IL-20, IL-26), TNFα and IFNγ, and optionally the plurality of S / D / M factors include IL-10R activator, IL-4, TNFα and IFNγ.

13. The method according to any one of claims 1 to 9, wherein the plurality of S / D / M factors further comprise a GM-CSF receptor (GM-CSFR) activator.

14. The method according to claim 13, wherein the GM-CSFR activator is selected from the group consisting of GM-CSF, GM-CSFR agonist antibodies, and small molecule activators of GM-CSFR.

15. The method according to claim 14, wherein the GM-CSFR activator is GM-CSF.

16. The method according to any one of claims 1 to 9, wherein the plurality of S / D / M factors further comprises an IL-6 receptor (IL-6R) activator, and optionally the IL-6R activator is selected from the group consisting of IL-6, IL-6R agonist antibodies, and small molecule activators of IL-6R.

17. The method according to claim 16, wherein the IL-6R activator is IL-6.

18. The method according to any one of claims 1 to 9, further comprising the step of contacting the population of monocytes with a plurality of refining factors selected from the group consisting of type I interferon, IFNγ, TNFα, TLR ligand, CD40L or CD40 ligation antibody, anti-PD-L1 antibody and TPI-1, wherein the type I interferon optionally comprises IFNα and / or IFNβ, and the TLR ligand optionally comprises polyIC, CpG or LPS.

19. The method according to claim 18, wherein the refining factor comprises IFNα, IFNγ, and TNFα.

20. The method according to claim 19, wherein the refining factor further comprises at least two agents selected from the group consisting of polyIC, CpG, CD40L, R848, and an anti-PD-L1 antibody, and optionally the refining factor comprises an SHP-1 inhibitor (e.g., TPI-1).

21. A method for promoting the survival of a population of monocytes derived from an individual in in vitro culture, comprising the step of culturing the population of monocytes in a medium having one or more molecules that promote IL-10 receptor (IL-10R) expression on the monocytes, wherein the individual has cancer.

22. A method for promoting the survival of a population of monocytes derived from an individual in in vitro culture, comprising the step of culturing the population of monocytes in a medium having an IL-10R activator, wherein the individual has cancer, and optionally the IL-10R activator is selected from the group consisting of IL-10, IL-10 family members, IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of downstream STAT3 of IL-10R, and optionally the IL-10R activator is IL-10.

23. A method for increasing the expression of IL-10 receptor (IL-10R) in a population of monocytes derived from an individual having cancer, comprising the step of contacting the population of monocytes with one or more agents selected from the group consisting of an IL-10R activator, a TNFR activator, and an IFNGR activator.

24. A method for promoting the survival of a population of monocytes derived from an individual in in vitro culture, comprising the step of culturing the population of monocytes in a medium containing IL-10, TNFα and IFNγ, wherein the individual has cancer.

25. A method for promoting the differentiation of a population of individual-derived monocytes into antigen-presenting cells ("APCs") in vitro culture, comprising the step of culturing the population of monocytes in a medium having one or more molecules selected from the group consisting of IL-4 receptor (IL-4R) activator, TNFα receptor (TNFR) activator, and interferon-γ (IFNγ) receptor (IFNGR) activator, wherein the individual has cancer.

26. A population of APCs produced by the method according to any one of claims 1 to 9 and 25.

27. ​​A population of antigen-presenting cells ("APCs") derived from an individual having cancer, wherein the APCs are a) MHC-I+ / high, MHC-II+ / high, and CD40+ / high, b) TLR2+ / high and / or STING+ / high, and c) LOX1+ / high and / or uPAR+ / high, and optionally the expression level of CD40 on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level on monocytes, M1 macrophages, M2 macrophages, and / or MoDCs.

28. A method for activating a population of immune cells, comprising the step of co-culturing the population of immune cells with a population of APCs according to claim 26, wherein the APCs are pre-loaded with one or more neoantigen peptides.

29. The method according to claim 28, wherein the method comprises the step of contacting the APC with a composition comprising a plurality of neoantigen peptides, and / or the APC is pre-incubated with the composition.

30. The method according to claim 28, wherein the immune cells are selected from the group consisting of PBMCs, tumor-infiltrating T cells (TILs), and T cells, and the immune cells are T cells if necessary, and the T cells are CD8 T cells and / or CD4 T cells if necessary.

31. The method according to claim 30, wherein the activation is performed at least three times, each co-culture takes at least about five days, and two of the three co-cultures do not contain an anti-CD3 antibody or an anti-CD28 antibody.

32. The method according to claim 28, wherein the population of immune cells and the antigen-presenting cells originate from the same individual.

33. The method according to claim 28, wherein the population of immune cells and the antigen-presenting cells do not originate from the same individual.

34. A population of activated immune cells obtained by the method of claim 28.

35. A composition for treating cancer in a patient, comprising the population of APCs described in Claim 26.

36. A composition comprising a plurality of survival, differentiation and / or maturation factors ("S / D / M factors"), wherein the plurality of S / D / M factors comprises 1) an IL-10 receptor (IL-10R) activator and 2) one or more agents selected from the group consisting of an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon-gamma (IFNγ) receptor (IFNGR) activator.

37. The composition according to claim 36, wherein the IL-10R activator is selected from the group consisting of IL-10, IL-10 family members, IL-10R agonist antibodies, small molecule activators of IL-10R, and activators of downstream STAT3 of IL-10R.

38. The composition according to claim 37, wherein the IL-10R activator is IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, coliberin TFA, or galcinone D, and optionally the IL-10R activator is IL-10.

39. The composition according to claim 36, wherein the plurality of S / D / M factors comprises two or more agents selected from the group consisting of IL-4R activator, TNFR activator, and IFNGR activator.

40. The composition according to claim 39, wherein the plurality of S / D / M factors include IL-10, IL-4, TNFα, and IFNγ.

41. The composition according to any one of claims 36 to 40, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

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