Compositions and methods for activating immune cells

Abstract

The present invention provides compositions and methods for generating antigen presenting cells (APCs) from monocytes (e.g., monocytes from cancer patients) that relate to IL-10 receptor activators (e.g., IL-10), IFN gamma receptor activators (e.g., IFN gamma), TNF alpha receptor activators (e.g., TNF alpha), IL-4 receptor activators (e.g., IL-4), GM-CSF receptor activators (e.g., GM-CSF receptor activators). E.g., GM-CSF (e.g., GM-CSF) and / or an IL-6 receptor activator (e.g., IL-6). Also provided are APCs thus produced, as well as methods of activating immune cells (e.g., T cells) by co-culture with APCs. Also provided are activated immune cell compositions and methods of treatment involving activated immune cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 325,439, filed on March 30, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Field of the Invention

[0003] The present invention relates to compositions and methods for: a) promoting the survival of monocytes, b) promoting the differentiation and / or maturation of antigen presenting cells (APCs) derived from monocytes (such as monocytes from cancer patients or healthy donors), and c) activating immune cells (such as T cells). Background of the Invention

[0005] Professional antigen presenting cells (APCs) (e.g., dendritic cells (DCs) and macrophages) and the network of immunogenic immunity they mediate are central components in establishing antigen-specific acquired immunity to protect the host from cancer mutations, infectious pathogens, and tissue damage. In cancer immunotherapy, the promise of APCs is their ability to phagocytose tumor cells and present antigens to activate tumor-specific acquired immunity (including tumor-killing T cells and long-lasting anti-cancer antibodies). In addition, the successful preparation of APCs will also facilitate the development of APC vaccines to treat infections caused by viruses, bacteria, and other pathogens. Using these capabilities, once APC-based therapies are established, it is expected that complete cancer cures will be achieved to achieve systemic elimination of tumors and metastases, and to establish immune memory to prevent tumor recurrence / relapse.

[0006] However, the use of APCs to treat cancer has revealed some serious limitations in the past few years. For example, the isolation and ex vivo stimulation of autologous APCs have been proven to be time-consuming and expensive, and the quality of DCs produced in vitro is unstable. Therefore, the use of patient-derived autologous DCs will limit the standardization of DC-based treatment regimens. See Eggermont et al., Trends Biotechnol. 2014 Sep; 32 (9): 456-65. Among them, one of the most critical obstacles to APC therapy is the lack of effective technical means to differentiate monocytes from cancer patients into pro-inflammatory DC / macrophage-like efficient APCs.

[0007] The contents of all publications, patents, patent applications, and published patent application publications mentioned herein are incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0009] In one aspect, the present application provides a method for stimulating a monocyte population derived from an individual to produce an antigen presenting cell (APC) population, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 1) an IL-10 receptor (IL-10R) activator, and 2) one or more agents selected from the following group: an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon γ (IFNγ) receptor (IFNGR) activator. In some embodiments, the IL-10R activator is selected from: IL-10 (e.g., PEGylated IL-10, e.g., pegilodecakin 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 STAT3 downstream of IL-10R (e.g., long noncoding RNA (LncRNA) PVT1, NEAT1, FEZF1-AS1, UICC). See 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, the IL-10 is present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (eg, about 20 ng / ml).

[0010] In some embodiments according to any of the above methods, the plurality of S / D / M factors include an IL-4R activator, optionally wherein 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-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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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-13 is present in the culture medium at a concentration of at least about 30 pg / ml, optionally at least about 60 pg / ml, further optionally about 60 pg / ml to about 2 ng / ml (e.g., about 100 pg / ml to about 2 ng / ml).

[0011] In some embodiments according to any of the above methods, the plurality of S / D / M factors comprises a TNFR activator, optionally wherein the TNFR activator is selected from: 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 culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml).

[0012] In some embodiments according to any of the above methods, the multiple S / D / M factors include an IFNGR activator, optionally wherein the IFNGR activator is selected from: 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 culture medium at a concentration of at least about 5ng / ml, optionally at least about 10ng / ml, and further optionally about 10ng / ml to about 200ng / ml (e.g., about 50-100ng / ml).

[0013] In some embodiments according to any of the methods above, the plurality of S / D / M factors are present in a single composition.

[0014] In some embodiments according to any of the methods above, at least one of the plurality of S / D / M factors is provided separately from one of the other S / D / M factors in the plurality of S / D / M factors.

[0015] In some embodiments according to any of the above methods, 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. In some embodiments, the plurality of S / D / M factors comprises IL-10, IL-4, TNFα, and IFNγ.

[0016] In some embodiments according to any of the above methods, 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, 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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).

[0017] In some embodiments according to any of the above methods, the multiple S / D / M factors also include an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from: 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 human IL-6 or human recombinant IL-6. In some embodiments, the IL-6 is present in the culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, e.g., about 30 pg / ml).

[0018] In some embodiments according to any of the above methods, the multiple S / D / M factors are derived from T cell cultures treated with anti-CD3 antibodies and anti-CD28 antibodies, and optionally the multiple S / D / M factors are derived from the supernatant of the culture. In some embodiments, the T cells are isolated from PBMCs of the same individual or different individuals. 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 not pre-treated with anti-CD3 antibodies and / or anti-CD28 antibodies before treatment. In some embodiments, the T cells have been pre-treated with anti-CD3 antibodies and / or anti-CD28 antibodies before treatment. In some embodiments, the multiple S / D / M factors are derived from cultures after the T cells are treated with anti-CD3 antibodies and anti-CD28 antibodies for about 1-3 days (optionally about 2 days).

[0019] In some embodiments according to any of the methods above, the monocytes are cultured for at least about 2 days (e.g., about 2-4 days, about 2-3 days, about 2 days) in the presence of the S / D / M factors or medium derived from T cell culture.

[0020] In some embodiments according to any of the above methods, the method further comprises contacting the monocyte population with a plurality of expansion optimization factors, wherein the expansion optimization factor is selected from: type I interferon, IFNγ, TNFα, TLR ligand, CD40L or CD40-linked antibody, anti-PD-L1 antibody and TPI-1, optionally wherein the type I interferon includes IFNα and / or IFNβ, and optionally wherein the TLR ligand is poly IC, CpG or LPS. In some embodiments, the plurality of optimization factors are provided after the plurality of monocytes are contacted with the plurality of S / D / M factors or a medium derived from T cell culture, thereby producing the APC population, and wherein the APC population is cultured for about 1-5 days in the presence of the plurality of optimization factors, optionally wherein the APC population is cultured for about 1 day. In some embodiments, the multiple optimization factors are provided when: a) the monocytes are at least about 50% viable, b) at least about 30% of the APC population displays dendritic cell morphology, and / or c) the APC population 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. In some embodiments, the optimization factors include IFNα, IFNγ, and TNFα. In some embodiments, the optimization factors also include poly IC, CpG, CD40L, and anti-PD-L1 antibodies.

[0021] The present application provides, on the other hand, a method for promoting the survival of a monocyte population derived from an individual in an in vitro culture, comprising culturing the monocyte population in a culture medium containing one or more molecules that promote the expression of IL-10 receptor (IL-10R) on monocytes. In some embodiments, the one or more molecules include an IL-10R activator, optionally wherein the IL-10R activator is selected from: IL-10, an IL-10R agonist antibody, and a small molecule activator of IL-10R, and further optionally the IL-10R activator is IL-10.

[0022] On the other hand, the present application provides a method for promoting the survival of a monocyte population derived from an individual in an in vitro culture, comprising culturing the monocyte population in a culture medium containing an IL-10R activator, optionally wherein the IL-10R activator is selected from: IL-10, an IL-10R agonist antibody and a small molecule activator of IL-10R, and further optionally the IL-10R activator is IL-10.

[0023] In some embodiments according to the above methods of promoting survival of a monocyte population, the monocyte population expresses low levels of IL-10R prior to contacting with the molecule.

[0024] In some embodiments according to the above method of promoting the survival of a monocyte population, 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: TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, and optionally wherein the IFNGR activator is selected from: IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally the culture comprises TNFα and / or IFNγ.

[0025] In another aspect, the present application provides a method for increasing the expression of IL-10 receptor (IL-10R) in a monocyte population from an individual suffering from cancer, comprising contacting the monocyte population with one or more agents selected from the group consisting of an IL-10R activator, a TNFR activator, and an IFNGR activator.

[0026] In another aspect, the present application provides a method for promoting the survival of a monocyte population derived from an individual in an in vitro culture, comprising culturing the monocyte population in a culture medium containing IL-10, TNFα and IFNγ.

[0027] The present application provides, on the other hand, a method for promoting the differentiation of a monocyte population from an individual into an antigen presenting cell (APC) in an in vitro culture, comprising culturing the monocyte population in a culture 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 γ (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.

[0028] In some embodiments according to any of the methods above, the plurality of monocytes are obtained from peripheral blood of the individual, optionally wherein the monocytes express CD 14, wherein the monocytes are obtained from the peripheral blood.

[0029] In some embodiments according to any of the above methods, the individual has cancer. In some embodiments, the individual has advanced cancer. In some embodiments, the individual has a solid tumor.

[0030] In some embodiments according to any of the methods above, the individual has an inoperable tumor and / or metastasis.

[0031] In some embodiments according to any of the methods above, the individual is a human.

[0032] In another aspect, the present application provides an APC population produced by any of the above methods for producing an APC population. In some embodiments, the APC expresses a low level of an inhibitory signal molecule, wherein the inhibitory signal molecule is selected from: TGFβR, SIRPα, LilRBs and Siglec 10.

[0033] The present application provides an APC population on the other hand, wherein the APC expresses higher levels of one or more antigen presenting molecules compared to dendritic cells obtained from healthy people and cultured with GM-CSF and IL-4 for about 5 days, wherein the antigen presenting molecule is selected from: MHCI, MHCII, CD86, CD80, OX40L, ICAML, ICOSL and CD40, optionally wherein the APC is produced from monocytes in ex vivo cell culture, further optionally wherein the monocytes are obtained from cancer patients. In some embodiments, the APC expresses low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from: TGFβR, SIRPα, LilRBs and Siglec 10.

[0034] The present application provides a method of activating an immune cell population on the other hand, comprising co-culturing the immune cell population with the APC population of any one of the above-mentioned APC populations, wherein the APC is preloaded with one or more new antigenic peptides. In some embodiments, the method includes contacting the APC with a composition comprising a plurality of new antigenic peptides, and / or the APC has been pre-cultured with the composition. In some embodiments, the composition comprising a plurality of new antigenic peptides is a surgical resection of a tumor tissue, or a biopsy extract thereof. In some embodiments, the composition comprising a plurality of new antigenic peptides is a mixture of tumor cells, or an extract thereof separated from a tumor tissue or a biopsy. In some embodiments, the composition comprising a plurality of new antigenic peptides is a mixture of separated new antigenic peptides. In some embodiments, the separated new antigenic peptides are synthetic peptides. In some embodiments, the APC is allowed to contact the composition comprising a plurality of new antigenic peptides for about 4 hours to about 24 hours. In some embodiments, the immune cell is selected from: PBMC, tumor infiltrating T cells (TIL) and T cells, optionally the T cells are CD8 T cells and / or CD4 T cells. In some embodiments, the co-culture is performed for at least 24 hours. In some embodiments, the method further comprises expanding the immune cell population after the co-culture step. In some embodiments, expanding the immune cell population comprises contacting the immune cell 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 immune cell population and the antigen-presenting cell are derived from the same individual. In some embodiments, the immune cell population and the antigen-presenting cell are not derived from the same individual.

[0035] In another aspect, the present application provides an activated immune cell population obtained by any one of the above methods for activating an immune cell population.

[0036] In another aspect, the present application provides a method for treating cancer in a patient, comprising administering to the patient any of the above-mentioned APC populations and / or activated immune cells. In some embodiments, the APC or activated immune cells are administered intratumorally, intraperitoneally, or intravenously. In some embodiments, the activated immune cells are administered at about 10 7 Up to 10 9In some embodiments, the method further comprises treating the patient with chemotherapy, radiotherapy, 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 to be treated. In some embodiments, the APC or activated immune cell administered to the patient is derived from the patient. In some embodiments, the APC or activated immune cell administered to the patient is not derived from the patient. In some embodiments, the cancer to be treated is a solid tumor.

[0037] The present application provides, on the other hand, a composition comprising a plurality of survival, differentiation and / or maturation factors (S / D / M factors), wherein the plurality of S / D / M factors include: 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 includes an IL-4R activator, optionally wherein 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 multiple S / D / M factors include TNFR activators, optionally wherein the TNFR activators are selected from: TNFα, TNFR agonist antibodies, and small molecule activators of TNFR. In some embodiments, the TNFR activator is TNFα. In some embodiments, the multiple S / D / M factors include IFNGR activators, optionally wherein the IFNGR activators are selected from: IFNγ, IFNGR agonist antibodies, and small molecule activators of IFNGR. In some embodiments, the TNFR activator is IFNγ. In some embodiments, the multiple S / D / M factors include two or more preparations selected from the group consisting of: IL-4R activators, TNFR activators, and IFNGR activators. In some embodiments, the multiple S / D / M factors include IL-10, IL-4, TNFα, and IFNγ. In some embodiments, the multiple S / D / M factors also include GM-CSF receptor (GM-CSFR) activators. 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 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, IL-6R agonist antibodies, and small molecule activators of IL-6R. In some embodiments, the IL-6R activator is IL-6. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figures 1A-1EDescribe the failure of monocytes from cancer patients (cMo) to respond to the macrophage / DC-differentiation factors M-CSF and GM-CSF, which induce differentiation of monocytes from healthy donors (Mo). Figures 1A-1C showed that a high rate of cMo cell death was associated with differentiation failure. Figure 1C showed that the combination of tumor-conditioned medium partially protected cMo from cell death, while differentiation failure was induced by M-CSF plus GM-CSF. Figure 1D It was shown that cMo displayed reduced expression of MCSF-R and GMCSF-R compared to Mo. Figure 1E Various combinations of reagents were shown to either support cMo survival or fail to differentiate.

[0039] Figure 2A-2C Different signaling events occurred in Mo derived from healthy individuals relative to cancer patients in response to M-CSF and GM-CSF. Figure 2A It is shown that M-CSF and GM-CSF: 1) trigger activation of Akt and Erk1 / 2 in Mo but not cMo, and 2) trigger cleavage of caspase-9 and caspase-3 in cMo but not Mo, as evidenced by increased phosphorylation of both Akt and Erk1 / 2 as analyzed by Western blotting. Figure 2B Indicating that as a result of cell apoptosis, cMo cell survival % was significantly reduced compared to Mo cell survival % . Figure 2C Shown are the total levels of common intracellular signaling proteins, including Akt and Erk1 / 2, which were not reduced in cMo cells compared to Mo.

[0040] Figures 3A-3I Differentiation of monocytes (cMo) from cancer patients using TCR-activated T cell conditioned medium (Karnelian X1 or Carnelian X1) is described. Figure 3A Describe the experimental protocol. Figures 3B to 3C The levels of cytokines produced from the first round of TCR stimulation of CD4 T cells derived from individuals with autoimmune disorders are shown, and the results confirm high levels of IL-10 production. Figure 3D Shown are the cytokine profiles of CD4 and CD8 T cells derived from individuals with autoimmune disorders after the first and second rounds of TCR stimulation. Figure 3E and 3F Shown are the morphologies of cMo from patient SD-21-451 treated with activated T cell medium or M-CSF. Figure 3F It was shown that cMo differentiation was induced by medium conditioned by TCR-activated T cells. Figure 3Gand 3H Shown are the morphology and cell surface expression of antigen presentation machinery in cMO from patient SD-21-451 after 72 h of differentiation by Carnelian X1, indicating that Carnelian X1 differentiates cMO into professional APCs. Fig. 3I Indicates the percentage of cMo that survived and differentiated into κAPCs, which were strongly supported using medium with high IL-10 concentration (ie, KX1 medium), but not in medium of healthy CD4 Te cells lacking IL-10.

[0041] Figure 4 Characterize how monocytes from healthy donors or cancer patients survive and differentiate under different conditions (e.g., M-CSF, GM-CSF, or Carnelian X1).

[0042] Figures 5A-5K Describe the important components in Carnelian X1. Figure 5A Shows cytokines produced by T cells after TCR stimulation. The culture medium collected on day 2 (48 hours after stimulation) is called Carnelian X1. Figure 5B Cytokine depletion analysis identified IL-10, IFNγ, and TNFα in carnelian X1 as important for cMo survival. Figure 5C IL-10 (recombinant) was shown to dose-dependently restore IL-10-depleted Carnelian X1 for supporting cMo survival. Figure 5D Shown are the % cMo survival when treated with the JAK inhibitor C188-9 or the STAT3 inhibitor Napabucasin. cMo survival was decreased in a dose-dependent manner. Figure 5E It was shown that the STAT3 activators Colivelin TFA and Garcinone D both restored cMo survival in a dose-dependent manner in cMo cells grown in medium with low IL-10 concentrations. Fig. 5F Depicts the cMo survival % of cells cultured with medium 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. Figure 5G cMo survival % is depicted for cells cultured with medium containing IL-12 family member cytokines (ie, IL-12 and IL-23), following a similar pattern but treated with lower levels of IL-10, thereby restoring cMo survival. Figure 5H It was shown that IL-12 treatment induced IL-10 production by cMo. Fig.5I Provided are graphical representations summarizing cMo survival and differentiation into κAPC in response to treatment with specific cytokines or STAT3 activators. Figure 5J Shown is the % survival of cMo when treated with IL-6 or IL-11 in low IL-10 medium, which was significantly reduced compared to cMo cultured in medium with IL-10. Figure 5K Shown is the % survival of cMo when treated with G-CSF in low IL-10 medium, which was significantly reduced compared to cMo cultured in medium with IL-10.

[0043] Figures 6A-6G The phenotypes of cMo grown in medium supplemented or lacking specific cytokines are described. Fig. 6A We show that cytokine depletion analysis identified IL-4, IFNγ, and TNFα as being important for cMo phenotypic differentiation into APCs, and that depletion of IFNγ, TNFα, or IL-4 each delayed cMo to APC differentiation, as indicated by decreased expression of MHC-II, CD80 / 86, and CD40. Figure 6B and 6D The cytokine cocktail (C-combo) shown recapitulates Carnelian X1 for supporting cMo survival and APC differentiation. Figure 6C Shown are the effects of KX1 treatment on cMo derived from patients with different stages of liquid or solid cancer. Figures 6E to 6G An overview of the components in carnelian X1 that support cMo to APC differentiation within 1 and 2 days, respectively, is shown.

[0044] Figures 7A-7C Describe the mechanism by which carnelian X1 induces cMo survival and differentiation into APCs. Figure 7A-7B Shown are cytokine receptor expression on cMo before and after carnelian X1 treatment compared with Mo from healthy donors. Figure 7C Hypothesis describing cytokine-mediated upregulation of IL-10R leading to cMo survival followed by GM-CSF, TNFα, IL-6 and IFNγ-mediated phenotypic differentiation into immunogenic APCs.

[0045] Figures 8A-8C KX1 activated PI3K-Akt, MAPK, and STAT3 cell survival pathways and protected against apoptosis mediated by caspase-1. Fig. 8A showed that KX1 induced activation of Akt, Erk1 / 2, and STAT3 into cMo, whereas both M-CSF and GM-CSF failed, as confirmed by protein phosphorylation levels assessed by Western blotting. Figure 8B It was demonstrated that KX1 treatment prevented cMo from undergoing apoptosis as indicated by the levels of cleaved caspase-9 and caspase-3 measured by Western blotting. Figure 8CThe results showed that the PI3K-Akt and MAPK pathways were involved in the regulation of KX1-mediated cMo survival, such that inhibition of PI3K, Akt or MAPK reduced cMo survival, whereas inhibition of NFκB had no effect on cMo survival.

[0046] Figures 9A-9D Shown, IL-10 induced survival signaling in cMo. Figures 9A-9B cMo cultured without treatment (NT), with KX1 or with KX1 supplemented with depletion of individual cytokine components are shown. 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. Fig. 9C Shown is the time course activation of Akt, Erk1 / 2, and STAT3 as measured by protein phosphorylation levels in cMo grown in KX1 with or depleted of IL-10. Fig.9D Show from Fig. 9C Densitometry results calculated from acquired Western blot images.

[0047] Figures 10A-10D Displays C-combo component analysis. Figures 10A-10C Describes C-combo V1-V4, which were designed to test the minimal compositional requirements for activation of Akt, Erk1 / 2, and STAT3 in cMo ( Figures 10A-10B ) and supports the differentiation of cMo into κAPC ( Fig. 10C ). Fig. 10D High IL-10 KX1 medium was compared with C-combo and similar activation of Akt, Erk1 / 2 and STAT3 in cMo was found.

[0048] Fig.11 The phenotype of κAPCs differentiated from cMo derived from cancer patients was characterized and the expression of antigen presentation machinery of cells grown in KX1 with IL-10 supplementation or in C-combo medium was compared.

[0049] Figures 12A-12H Describes the optimization of APC phenotype by Carnelian reagent ( Figures 12A-12B :Opt1-4; Figures 12C-12D :Opt 1-5). Fig. 12A Shown are the phenotypes of APCs differentiated from cMo from cancer patients after a) treatment with Carnelian X1 for 48 hours and b) treatment with Opt1, 2, 3 or 4 for an additional 24 hours. Fig. 12B Opt 3 identified in. Fig. 12C Shown are: a) the phenotype of APCs differentiated from cMo of cancer patients after treatment with Carnelian X1 for 48 hours and b) Opt 1, 2, 3, 4 or 5 for an additional 24 hours. Fig.12D Instructions, Kanelian X2 is Opt 5. Fig.12E Describes the determination of the pro-inflammatory κAPC phenotype by detecting inflammatory cytokines secreted by κAPC into the cell-free culture medium. Fig.12F An assay of κAPC phagocytosis of tumor fragments is described, in which CFSE-stained tumor cells are snap-frozen in liquid N2, then freeze-thawed on ice, and tumor cell fragments are added to κAPC cultures in KX2Opt 5. Figure 12G Analysis of cell surface inhibitory receptors on kappa APCs is described, which confirmed low receptor content, with the exception of increased expression identified for LILRB1 and LILRB3. Inhibition of SHP-1 by TPI-1 was used to enhance proinflammatory responses. Fig.12H Examples of enhanced pro-inflammatory responses to TPI-1 treatment are described.

[0050] Figures 13A-13B The protocol for κAPC production using KX1 and KX2 is shown. Fig.13A Description of Carnelian X1 and X2 reagents manufactured from cMo K Overview of APC. Kanelian X1 can be replaced by C-combo. Fig. 13B Describe the surface expression of various molecules on fresh cMo and cMo-derived APCs, following culture with Carnelian X1 and Carnelian X2.

[0051] Fig.14A Show that the use of KX1 and / or KX2, and immature DC, mature DC, M0 macrophages M1 and M2 Compared to the cellular morphology of κAPCs assessed by light microscopy; and

[0052] Fig. 14B Shown is the size of κAPC compared to other cell types.

[0053] Figures 15A-15B Characterize the antigen presenting capacity of cMo-derived κAPCs and compare healthy Mo-derived κAPCs with MoDCs and M1 and M2 Compare. Fig.15A Showing that healthy Mo-derived κAPCs are different from MoDCs and M1 and M2 comparison. Fig. 15B Shown is the level of antigen presenting capacity of cMo derived from patients with advanced inoperable prostate cancer, colorectal cancer, or prostate cancer, as assessed by flow cytometry.

[0054] Figures 16A-16CShown are gene signatures from Nanostring transcriptional profiling of Mo, cMo, Mo-derived κAPC, and cMo-derived κAPC. Fig.16A Describe the relationship between mature MoDC treated with LPS and treated with LPS and IFNγ Gene signatures of Mo or cMo compared with those of κAPCs derived from healthy individuals and cancer patients. Fig. 16B Transcriptional analysis of Mo, cMo, Mo-derived κAPC, and cMo-derived κAPC is shown and demonstrates that Mo and cMo share similar gene signatures, however some differences were identified in the Mo-derived κAPC and cMo-derived κAPC gene signatures. Fig. 16C Transcriptional analysis of cMo-derived κAPC grown in KX1 medium versus C-combo medium, where the gene signature was similar, is provided.

[0055] Fig.17 Describe the paired comparisons in the transcription of the following genes: 1) Mo-derived κAPC relative to Mo, Mo-derived κAPC relative to M1 and Mo-derived κAPCs versus MoDCs stimulated with LPS; 2) cMo-derived κAPCs versus cMo, and C-combo versus KX1 medium; and 3) Mo versus cMo, and Mo-derived κAPCs versus cMo-derived κAPCs.

[0056] Figures 18A-18B Description and DC or Expression of different cell surface markers on κAPC compared to κAPC. Fig.18A Describe the different DC subtypes, Mo-derived DC (MoDC), M1 and cell surface markers found on κAPC. Fig.18B Show in Fig.18A Flow cytometric analysis of cell surface markers identified in the κAPCs confirmed that κAPCs did not associate with cDC1, cDC2, or pDCs or with MoDC or M1 Share the same spectrum.

[0057] Figures 19A-19C Provides identification of κAPC-specific cell surface markers. Fig.19A Showing that κAPCs are related to MoDCs and M1 Comparison of cell surface expression between , where cell surface proteins are specifically increased on κAPCs (ie, IL-3R and CD32; indicated with asterisks). Fig.19B Display of κAPC and MoDC, M1 and M2 Comparison of cell surface proteins between MoDC and M1 and M2 In contrast, Trem2, C3AR, IL-3R, LOX1, uPAR, CD40, and TLR2 were increased on κAPCs. Fig.19C Display and M1 In comparison, pattern recognition receptors expressed by κAPC were expressed by κAPC, with κAPC showing much higher levels of STING and TLR2.

[0058] Fig. 20 To describe the combination therapy of kappa APC and tumor-focused RT for KPC pancreatic ductal adenocarcinoma.

[0059] Figures 21A-21B Describing therapeutic TT against pancreatic cancer K APC vaccine. Establishment of syngeneic KPC pancreatic ductal adenocarcinoma by subcutaneous implantation. Fig.21A Describes the use of intratumoral (IT) injection of TT- K Experimental schedule and tumor volume changes in APC-vaccinated mice. Fig. 21B TME analysis revealed that TT-inoculated K In APC vaccine-treated tumors, there was a significant increase in CD45+ immune cells, especially the CD8 T cell population.

[0060] Figures 23A-23D To characterize NeoT cell targeting and clearance of multiple myeloma (MM) in vitro. Fig.23A The figure shows the expansion level of NeoT cells after stimulation and activation with κAPC or anti-CD3 / anti-CD28 antibodies. Fig. 23B Provided are light microscopy images of NeoT expansion following co-culture with MM antigen-loaded κAPCs. Fig.23C MM-specific NeoT cells that exhibit potent cytolytic activity against autologous MM cells in total bone marrow but avoid non-bone marrow cells are described. NeoT activated by anti-CD3 / anti-CD28 antibody stimulation showed limited cytolytic effect against MM cells. Fig.23D Still images from a time-lapse video of NeoT cells killing MM cells in in vitro co-culture are provided.

[0061] Figures 24A-24C κAPC activation of NeoT cells derived from ovarian cancer tumor-infiltrating lymphocytes (TILs) is described. Fig.24A NeoT cell proliferation following activation by κAPC or anti-CD3 / anti-CD28 antibody stimulation is shown, where the NeoT continuously responds to κAPC activation but fails to respond to a second round of anti-CD3 / anti-CD28 antibody stimulation. Fig. 24BComparison of CD4 and CD8 T cell numbers in TILs before adding κAPCs to in vitro cultures, and then after 10 days of culture with κAPCs. After 10 days of expansion, NeoT cells showed significant in vitro expansion. Fig.24C Provided are light microscopy images of NeoT cells targeting in vitro dissociated ovarian cancer cells for cytolysis, which were recorded by time-lapse video recording. The graphical representation of the percentage of ovarian cancer cells lysed shows that the tumor-specific cytotoxicity of κAPC-activated Neo-T cells was significantly increased.

[0062] Fig.25 The activation of kappa APCs by autologous NeoT cells derived from TILs of various solid tumors (ie, liver metastatic urothelial carcinoma, ovarian, colon, renal, and lung cancer) is described.

[0063] Fig.26 Autologous NeoT cells that engage kappa APCs and are then activated and clonally expanded in vitro are described.

[0064] Figures 27A-27C To describe the development of Neo-T therapy for patients with metastatic uterine leiomyosarcoma. Fig.27A Differentiation of κAPCs from autologous PBMC mononuclear cells (cMo) using Carnelian X1 and Carnelian X2 is shown. κAPCs show potent antigen presenting ability. Fig.27B Showing the production of Neo-T in vitro. Fig.27C Neo-T is effective in killing cancer cells. 7 Neo-T cells were administered intratumorally (it) into liver metastatic masses (upper panel), and the treatment induced a reduction in tumor volume, as assessed after 4 weeks (lower panel).

[0065] Figures 28A-28B Describes the changes in tumor volume in C57Bl / 6 mice implanted with KPC pancreatic ductal adenocarcinoma and given NeoT adaptive cell therapy ( Fig.28A ) and overall survival rate ( Fig.28B ) measured tumor progression. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present application provides novel compositions and preparations for converting a large number of monocytes, particularly monocytes from cancer patients, into potent antigen presenting cells (hereinafter referred to as "APCs"). These APCs can then be used to activate immune cells, making them highly effective therapeutic agents for cancer treatment.

[0068] The inventors have discovered that monocytes from cancer patients (referred to as "cMo" or "cMos") are unable or inefficiently differentiated into dendritic cells or macrophage-like APCs by traditional methods (e.g., stimulation with M-CSF and / or GM-CSF). This inhibition can unexpectedly be "unlocked" by cell culture supernatants of activated T cells containing high levels of IL-10. The inventors have unexpectedly discovered that IL-10, which is generally believed to be immunosuppressive in nature, is necessary to remove the immunosuppressive state of cMo. Without being limited to theory, it appears that IL-10 provides 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 that IL-4, IFNγ, and TNFα in the supernatant further promote differentiation from monocytes into APCs. Based on these profound findings, the inventors created a composition comprising one or more of these key factors from scratch, comprising an IL-10 receptor activator (such as IL-10) together 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, and confirmed that this composition can achieve the same results initially observed with the supernatant. When loaded with tumor-associated peptides (tumor cells / antigens), the APCs were shown to present antigens and activate tumor antigen-specific CD4 and CD8 T cells, making them particularly effective in treating cancer.

[0069] Therefore, the present application provides methods for generating APCs from monocytes (such as cMo), uses of APCs to activate immune cells, and uses of activated immune cells in treating cancer. Compositions comprising the key factors discussed herein are also provided.

[0070] Therefore, the present application provides, in one aspect, a method for stimulating a monocyte population from an individual to produce an antigen presenting cell (APC) population, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 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. The S / D / M factors do not need to exhibit the same function.

[0071] In another aspect, the present application provides a population of APCs (such as APCs produced by some of the above methods), and uses of APCs for cancer treatment.

[0072] The present application provides a method for activating an immune cell population on the other hand, comprising co-culturing the immune cell population (e.g., T cells) with an APC population as described herein, wherein the APC is pre-loaded with one or more new antigenic peptides. Also provided is an activated immune cell population obtained using the method, and a method for treating cancer by administering the activated immune cells.

[0073] On the other hand, the present application provides a composition comprising 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, and 2) one or more agents selected from the following group: an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon γ (IFNγ) receptor (IFNGR) activator.

[0074] I. Definitions

[0075] In general, the terms used in the claims and this specification are intended to be interpreted as having the ordinary meaning understood by those skilled in the art. Certain terms are defined below to provide additional clarity. In the event of a conflict between the ordinary meaning and the provided definition, the provided definition shall apply.

[0076] The terms "subject," "subject," or "patient" are used synonymously herein to describe mammals, including humans. Subjects include, but are not limited to, humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is a human. In some embodiments, the subject has a disease, such as cancer. In some embodiments, the subject is in need of treatment.

[0077] As used herein, "monocyte" and "cell" should be understood to refer not only to the monocyte or cell when obtained, but also to the progeny or potential progeny of the cell. Because certain modifications may occur in the progeny due to, for example, 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.

[0078] When referring to the expression of a surface molecule in a cell population (e.g., monocytes or APCs), "high level" or "higher level", "low level" or "lower level" refers to how the average expression level of a specific surface molecule on the cell population is compared to the average level of the surface molecule on a reference cell population. Unless otherwise specified, a reference cell population refers to a corresponding cell population derived from a healthy donor. In some cases, when the expression level of a specific molecule on the 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, the molecule is defined as a high level. In some cases, when the expression level of a specific molecule on the 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, the molecule is defined as a low level.

[0079] As used herein, "reference" refers to any sample, standard or level used for comparison purposes. The reference can be obtained from a healthy and / or non-diseased sample. In some instances, the reference can be obtained from an untreated sample. In some instances, the reference is obtained from a non-diseased or untreated sample of an individual. In some instances, the reference is obtained from one or more healthy individuals who are not the individual or patient.

[0080] As used herein, the term "antigen" is a substance that induces an immune response.

[0081] As used herein, the term "neoantigen" is an antigen with at least one change that makes it different from the corresponding wild-type parent antigen, for example, by mutation of tumor cells or post-translational modification specific to tumor cells. The new antigen may comprise a polypeptide sequence. Mutations leading to new antigens may include frameshift or non-frameshift insertions and deletions (indels), missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes leading to neoORFs. Mutations may also comprise splice variants. Post-translational modifications specific to tumor cells may include abnormal phosphorylation. Post-translational modifications specific to tumor cells may also include spliced ​​antigens produced by the proteasome. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. October 21, 2016; 354(6310): 354-358.

[0082] As used herein, the term "tumor neoantigen" or "cancer neoantigen" is a neoantigen that is present in tumor cells or tissues of an individual, but not in the corresponding normal cells or tissues of the individual.

[0083] The term "peptide" refers to a polymer of amino acids (including protein fragments) of no more than 100 amino acids, which may be linear or branched, contain modified amino acids, and / or be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified naturally or by interference (including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification). Also included in this term are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and 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 synthesized (e.g., chemically synthesized or synthesized by recombinant DNA technology).

[0084] As used herein, the term "epitope" is a specific portion of an antigen that is usually bound by an antibody or T-cell receptor.

[0085] As used herein, the term "immunogenicity" is the ability to elicit an immune response, for example, by T-cells, B cells, or both.

[0086] As used herein, the terms "HLA binding affinity", "MHC binding affinity" mean the binding affinity between a specific antigen and a specific MHC allele.

[0087] As used herein, the term "HLA type" is the complement of HLA gene alleles.

[0088] As used herein, "activated T cells" refers to a population of monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., clones encoding different TCRs) T cells having a T cell receptor that recognizes at least one tumor antigen peptide. Activated T cells may contain one or more T cell subtypes, 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.

[0089] As used herein, "treatment" or "treating" is a method of obtaining a beneficial or desired result (including a clinical result). For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: reducing one or more symptoms arising from the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the occurrence or recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing regression of the disease (whether partial or complete), reducing the dose of one or more other agents required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. "Treatment" also includes a reduction in the pathological results of cancer. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0090] As used herein, "delaying" the development of cancer means postponing, hindering, slowing down, delaying, stabilizing and / or postponing the development of the disease. This delay may be a varying length of time, depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that sufficient or significant delay may actually include prevention, in that the individual does not develop the disease. A method of "delaying" the development of cancer is a method that reduces the likelihood of disease development within a given time frame and / or reduces the extent of the disease within a given time frame when compared to not using the method. These comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer development can be detected using standard methods, including, but not limited to: computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography or biopsy. Development may also refer to cancer progression that may not be initially detectable and includes occurrence, recurrence and onset.

[0091] As used herein, the term "simultaneous administration" means that the first therapy and the second therapy in the combination therapy are administered with a time interval of no more than about 15 minutes (e.g., no more than about 10 minutes, 5 minutes, or 1 minute). When the first therapy and the second therapy are administered simultaneously, the first therapy and the second therapy can be contained in the same composition (e.g., a composition comprising both the first therapy and the second therapy) or in separate compositions (e.g., the first therapy is in one composition and the second therapy is contained in another composition).

[0092] As used herein, the term "sequential administration" means that the first therapy and the second therapy of the combination therapy are administered with a time interval of more than about 15 minutes (e.g., more than about any of 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or more minutes). The first therapy or the second therapy can be administered first. The first therapy and the second therapy are contained in separate compositions, which can be contained in the same or different packages or sets.

[0093] As used herein, the term "administered in conjunction with" means that the administration of a first therapy and the administration of a second therapy in the combination therapy overlap with each other.

[0094] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or originally undesirable, for example, a material can be incorporated into a pharmaceutical composition administered to an individual without causing any significant undesirable biological effect, or without interacting in an adverse manner with any of the other components of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicology and manufacturing testing, and / or are included in the Inactive Ingredient Guide established by the US Food and Drug Administration.

[0095] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0096] Reference herein to "about" a value or parameter includes (and describes) variations to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0097] As used herein, reference to a "non" value or parameter generally means and describes "not" the value or parameter. For example, the method is not used to treat type X cancer, which means that the method is used to treat cancers of types other than X.

[0098] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0099] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0100] Any term not directly defined herein should be understood to have a meaning commonly associated therewith, as understood in the technical field of the present invention. Certain terms are discussed herein to provide additional guidance to practitioners when describing compositions, devices, methods, etc. of various aspects of the present invention and how to prepare or use them. It should be understood that more than one method can be used to describe the same thing. Therefore, alternative language and synonyms can be used in any one or more of the terms discussed herein. It is not important whether the term is elaborated or discussed herein. Some synonyms or alternative methods, materials, etc. are provided. Unless explicitly specified, the detailed description of one or more synonyms or equivalents does not exclude the use of other synonyms or equivalents. The use of examples (including examples of terms) is for illustrative purposes only, and does not limit the scope and meaning of aspects of the present invention herein.

[0101] II. Methods of Stimulating Monocyte Populations to Generate APCs

[0102] The present application provides various methods for stimulating a population of monocytes from an individual to generate a population of APCs.

[0103] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to promote survival of the monocytes is provided, comprising contacting the population of monocytes with an IL-10 receptor (IL-10R) activator (e.g., IL-10, e.g., IL-12).

[0104] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) an IL-10 receptor (IL-10R) activator (e.g., IL-10), and 2) one or more (e.g., two or more) agents 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 γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ). 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) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, and 2) two or more agents selected from the group consisting of: IL-4 receptor (IL-4R) activators (e.g., IL-4), TNFα receptor (TNFR) activators (e.g., TNFα) and interferon γ (IFNγ) receptor (IFNGR) activators (e.g., IFNγ). 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 one of the other S / D / M factors in 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 multiple S / D / M factors further include an IL-6 receptor (IL-6R) activator (e.g., IL-6). In some embodiments, in the presence of at least one of the S / D / M factors, monocytes are cultured for about 1-3 days (e.g., 2-3 days). In some embodiments, the multiple S / D / M factors are contained in a composition derived from a culture medium (e.g., supernatant) of a T cell culture treated with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is a CD8 T cell. In some embodiments, the T cell is isolated from the PBMC of the same individual or different individuals, and is not treated with anti-CD3 antibodies and / or anti-CD28 antibodies in advance before treatment. In some embodiments, the T cell is isolated from the PBMC of the same individual or different individuals, and is previously treated with anti-CD3 antibodies and / or anti-CD28 antibodies before treatment.In some embodiments, the culture medium is derived from a culture of T cells treated with anti-CD3 antibody and anti-CD28 antibody for about 1-3 days (optionally about 2 days).

[0105] In some embodiments, a method of stimulating a monocyte population derived from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of 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), wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, and 3) TNFα, wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-10R on monocytes 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, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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 monocyte population derived from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of 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), wherein at least the IL-4R activator is provided separately from the IL-10R activator or the TNFR activator. In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, and 3) TNFα, wherein at least IL-4 is provided separately from IL-10 or TNFα. In some embodiments, an IL-4R activator is provided after providing an IL-10R activator. In some embodiments, an IL-4R activator is provided after providing a TNFR activator. 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, the IL-10 is present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 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) prior to contact with an S / D / M factor. In some embodiments, the level of IL-4R on monocytes 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) prior to contact with an S / D / M factor.

[0107] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors comprise: 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) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, a method of stimulating a monocyte population derived from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, and 3) IFNγ, wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally 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 culture medium at a concentration of at least about 15pg / ml, optionally at least about 30pg / ml, and further optionally about 30pg / ml to about 1ng / ml (e.g., about 100pg / ml to about 1ng / ml). In some embodiments, the IFNγ is human IFNγ, or human recombinant IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5ng / ml, optionally at least about 10ng / ml, and further optionally about 10ng / ml to about 200ng / ml (e.g., about 50-100ng / ml). In some embodiments, the monocytes are cultured for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-10R on monocytes 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, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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, a method of stimulating a monocyte population derived from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of 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) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein at least the IL-4R activator is provided separately from the IL-10R activator or the IFNGR activator. In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, and 3) IFNγ, wherein at least IL-4 is provided separately from IL-10 or IFNγ. In some embodiments, after providing an IL-10R activator or IL-10, an IL-4R activator or IL-4 is provided. In some embodiments, after providing an IFNGR activator or IFNγ, an IL-4R activator or IL-4 is provided. In some embodiments, an IL-10R activator or IL-10 and an IFNGR activator or IFNγ are provided simultaneously. In some embodiments, an IL-10R activator or IL-10 and an 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 culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (eg, about 50-100 ng / ml).In some embodiments, the monocytes are cultured for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) before contact with the S / D / M factors. In some embodiments, the level of IL-4R on the monocytes 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) before contact with the S / D / M factors.

[0109] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors comprise: 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 γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, a method of stimulating a monocyte population derived from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 1) IL-10, 2) TNFα, and 3) IFNγ, wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml). In some embodiments, the monocytes are cultured for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-10R on monocytes 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, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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 of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of 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 γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein at least the IL-10R activator is provided separately from the TNFR activator or the IFNGR activator. In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) TNFα, and 3) IFNγ, wherein at least IL-10 is provided separately from TNFα or IFNγ. In some embodiments, an IL-10R activator or IL-10 is provided before providing a TNFR activator or TNFα. In some embodiments, an IL-10R activator or IL-10 is provided before providing an IFNGR activator or IFNγ. In some embodiments, a TNFR activator or TNFα and an IFNGR activator or IFNγ are provided simultaneously. In some embodiments, a TNFR activator or TNFα and an 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 culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 (eg, 2-3 days).In some embodiments, the level of IL-10R on monocytes 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) prior to contact with an S / D / M factor. In some embodiments, the level of IL-4R on monocytes 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) prior to contact with an S / D / M factor.

[0111] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of 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) a TNFα receptor (TNFR) activator (e.g., TNFα), and 4) an interferon γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, 3) TNFα, and 4) IFNγ, wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 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) prior to contact with an S / D / M factor. In some embodiments, the level of IL-4R on monocytes 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) prior to contact with an S / D / M factor.

[0112] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of 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) a TNFα receptor (TNFR) activator (e.g., TNFα), and 4) an interferon γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), wherein at least one of the plurality of S / D / M factors is provided separately from at least one of the other S / D / M factors in the plurality of S / D / M factors. 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) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, 3) TNFα, and 4) IFNγ, wherein at least one of the plurality of S / D / M factors is provided separately from at least one of the other S / D / M factors in the plurality of S / D / M factors. In some embodiments, IL-10R activator or IL-10 is provided before providing at least one of the other factors (e.g., IL-4R activator or IL-4). In some embodiments, IL-4R activator or IL-4 is provided after providing at least one of the other factors (e.g., IL-10R activator or IL-10, IFNGR activator or IFNγ, or TNFGR activator or TNFα). In some embodiments, IL-10R activator or IL-10, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided simultaneously. In some embodiments, IL-10R activator or IL-10, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided sequentially. In some embodiments, IL-4R activator or IL-4, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided simultaneously. In some embodiments, IL-4R activator or IL-4, TNFR activator or TNFα, and 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 culture medium at a concentration of at least about 2ng / ml, optionally at least about 10ng / ml, further optionally about 10ng / ml to about 200ng / ml (e.g., about 20ng / ml).In some embodiments, the IFNγ is human IFNγ, or human recombinant IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) prior to contact with the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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).

[0113] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of 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) a TNFα receptor (TNFR) activator (e.g., TNFα), 4) an interferon γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), and 5) one or two 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 the plurality of S / D / M factors are present in a single composition. 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) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, 3) TNFα, 4) IFNγ, and 5) one or two of GM-CSF and IL-6, wherein the plurality of S / D / M factors are present in a single composition. In some embodiments, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) prior to contact with the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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).

[0114] In some embodiments, a method of stimulating a monocyte population derived from an individual (e.g., a cancer patient) to generate an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of 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) a TNFα receptor (TNFR) activator (e.g., TNFα), 4) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), and 5) one or two 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 of the other S / D / M factors in the plurality of S / D / M factors. In some embodiments, a method of stimulating a population of monocytes from an individual (e.g., a cancer patient) to generate a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 1) IL-10, 2) IL-4, 3) TNFα, 4) IFNγ, and 5) one or two of GM-CSF and IL-6, wherein at least one of the plurality of S / D / M factors is provided separately from at least one of the other S / D / M factors in the plurality of S / D / M factors. In some embodiments, IL-10R activator or IL-10, and / or GM-CSFR activator or GM-CSF are provided before providing at least one of the other factors (e.g., IL-4R activator or IL-4, e.g., IL-6R activator or IL-6). In some embodiments, IL-4R activator or IL-4, and / or IL-6R activator or IL-6 are provided after providing at least one of the other factors (e.g., IL-10R activator or IL-10, e.g., GM-CSFR activator or GM-CSF). In some embodiments, IL-10R activator or IL-10, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided simultaneously. In some embodiments, IL-10R activator or IL-10, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided sequentially. In some embodiments, IL-4R activator or IL-4, TNFR activator or TNFα, and IFNGR activator or IFNγ are provided simultaneously. In some embodiments, IL-4R activator or IL-4, TNFR activator or TNFα, and 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 culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) prior to contact with the S / D / M factors.In some embodiments, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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).

[0115] In some embodiments, a method of stimulating a monocyte population from an individual (e.g., a cancer patient) to produce an antigen presenting cell (APC) population is provided, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 1) IL-10 (e.g., human IL-10 or human recombinant IL-10), 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), 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), wherein the plurality of S / D / M factors are present in a single composition. 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 culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) prior to contact with the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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).

[0116] In some embodiments, a method of stimulating a population of monocytes derived from an individual (e.g., a cancer patient) to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors comprise: 1) IL-10 (e.g., human IL-10 or human recombinant IL-10), 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), 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), wherein at least one of the plurality of S / D / M factors is provided separately from at least one of the other S / D / M factors in the plurality of S / D / M factors. In some embodiments, IL-10 and / or GM-CSF are provided before providing at least one of the other factors (e.g., IL-4 or IL-6). In some embodiments, IL-4 and / or IL-6 are provided after providing at least one of the other factors (e.g., IL-10 or GM-CSF). 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, the IL-10 is human IL-10, or human recombinant IL-10. In some embodiments, the IL-10 is present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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 for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on the monocytes 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) prior to contact with the S / D / M factors. In some embodiments, prior to contact with S / D / M factors, the level of IL-4R on monocytes 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).

[0117] In some embodiments, a method for stimulating a population of monocytes from an individual with cancer to produce a population of antigen presenting cells (APCs) is provided, comprising contacting the population of monocytes with a culture medium (e.g., supernatant) of T cells treated with anti-CD3 antibodies and anti-CD28 antibodies, wherein the culture medium comprises an IL-10R activator (e.g., IL-10). In some embodiments, the culture 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 culture 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 individual or different individuals, and are not pre-treated with anti-CD3 antibodies and / or anti-CD28 antibodies prior to treatment. In some embodiments, the culture medium is derived from a culture after treating T cells with anti-CD3 antibodies and anti-CD28 antibodies for about 1-3 days (optionally about 2 days). In some embodiments, monocytes are cultured for about 2-3 days in the presence of a culture medium derived from a T cell culture. 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 culture medium at a concentration of at least about 2ng / ml, optionally at least about 10ng / ml, and further optionally about 10ng / ml to about 200ng / ml (e.g., about 20ng / ml). In some embodiments, the IFNγ is human IFNγ, or human recombinant IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5ng / ml, optionally at least about 10ng / ml, and further optionally about 10ng / ml to about 200ng / ml (e.g., about 50-100ng / ml). In some embodiments, the TNFα is human TNFα, or human recombinant TNFα. In some embodiments, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, for example, 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally about 100 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 500 pg / ml, for example, about 300 pg / ml). In some embodiments, the monocytes are cultured for about 1-3 days (e.g., 2-3 days) in the presence of at least one of the S / D / M factors. In some embodiments, the level of IL-10R on monocytes 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) prior to contact with an S / D / M factor. In some embodiments, the level of IL-4R on monocytes 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) prior to contact with an S / D / M factor.

[0118] In some embodiments according to any one of the above embodiments, the method further comprises contacting the monocyte population with a plurality of optimization factors selected from the group consisting of type I interferon, IFNγ, TNFα, TLR ligand, CD40L or CD40-ligated antibody, 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, after contacting a plurality of monocytes with a plurality of S / D / M factors or a culture medium derived from a culture of T cells, a plurality of optimization factors are provided, thereby generating an APC population, and wherein in the presence of a plurality of optimization factors, the APC population is cultured for about 1-5 days, optionally wherein the APC population is cultured for about 1 day. In some embodiments, the plurality of optimizing factors is provided when: a) the monocytes are at least about 50% viable, b) at least about 30% of the APC population exhibits dendritic cell morphology, and / or c) the APC population 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.

[0119] In some embodiments, the method further comprises contacting the monocyte population with a plurality of optimizing factors including IFNα, IFNγ, and TNFα.

[0120] In some embodiments, the optimization factors include IFNα, IFNγ, TNFα, poly IC, CpG.

[0121] In some embodiments, the optimizing factors include IFNα, IFNγ, TNFα, poly IC, CpG, CD40L, and anti-PD-L1 antibodies.

[0122] In some embodiments, the optimizing factors include IFNα, IFNγ, TNFα, poly IC, CpG, CD40L, TPI-1, and anti-PD-L1 antibodies.

[0123] Survival, differentiation and / or maturation factors (S / D / M factors)

[0124] In some embodiments, the plurality of 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), and 2) one or more agents 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).

[0125] In some embodiments, the plurality of survival, differentiation and / or maturation factors (S / D / M factors) described herein include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (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 include: 1) IL-10 receptor (IL-10R) activators (e.g., IL-10), and 2) CpG.

[0126] In some embodiments, the plurality of 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).

[0127] In some embodiments, the plurality of 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), 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), 4) an IL-6 receptor (IL-6R) activator (e.g., IL-6), and 5) a GM-CSF receptor (GM-CSF) activator (e.g., GM-CSF).

[0128] In some embodiments, the plurality of 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).

[0129] In some embodiments, the plurality of survival, differentiation and / or maturation factors (S / D / M factors) described herein include: 1) a TNFα receptor (TNFR) activator (e.g., TNFα), 2) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), 3) an IL-6 receptor (IL-6R) activator (e.g., IL-6), and 4) a GM-CSF receptor (GM-CSF) activator (e.g., GM-CSF).

[0130] In some embodiments, the plurality of 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), 3) an interferon gamma (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), 4) an IL-6 receptor (IL-6R) activator (e.g., IL-6), 5) an IL-4 receptor (IL-4R) activator (e.g., IL-4), and 6) a GM-CSF receptor (GM-CSF) activator (e.g., GM-CSF).

[0131] IL-10 receptor (IL-10R) activators and IL-10

[0132] The "IL-10 receptor (IL-10R) activator" described herein refers to a molecule that activates the IL-10 receptor-mediated signal transduction pathway. IL-10R includes both IL-10R1 and IL-10R2.

[0133] 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 performs signal transduction through a receptor complex composed of two IL-10 receptor-1 proteins and two IL-10 receptor-2 proteins. Therefore, the functional receptor consists of four IL-10 receptor molecules. IL-10 binding induces STAT3 to perform signal transduction through the phosphorylation of JAK1 and Tyk2 through the cytoplasmic tails of IL-10 receptor 1 and IL-10 receptor 2. See, for example, Saraiva, M., O'Garra, A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 10, 170-181 (2010).

[0134] In humans, IL-10 is encoded by the IL10 gene, which is located on chromosome 1 and contains 5 exons, and is produced primarily by monocytes and, to a lesser extent, by lymphocytes (i.e., type II T helper cells (TH2), mast cells, CD4+CD25+Foxp3+ regulatory T cells, and activated T cells) and a subset of B cells. IL-10 can be produced by monocytes following PD-1 triggering in these cells.

[0135] IL-10 is a cytokine with multiple pleiotropic effects in immunomodulation and inflammation. It downregulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity and is involved in the regulation of the JAK-STAT signaling pathway.

[0136] Initially, IL-10 was reported to inhibit cytokine secretion, antigen presentation, and CD4+ T cell activation. Further studies have shown that IL-10 primarily inhibits lipopolysaccharide (LPS) and bacterial product-mediated induction of proinflammatory cytokines TNFα, IL-1β, IL-12, and IFNγ secretion from Toll-like receptor (TLR)-triggered myeloid cells.

[0137] The IL-10 mentioned herein includes any construct having an IL-10 component (e.g., naturally produced IL-10, e.g., recombinant IL-10). These include, but are not limited to, natural IL-10 (e.g., various subtypes of human IL-10), synthetic or recombinant IL-10, and fusion proteins having an IL-10 component.

[0138] In some embodiments, the IL-10R activator further comprises a moiety that enhances stability or half-life, including, for example, an Fc moiety or a PEG moiety.

[0139] In some embodiments, the IL-10R activator is selected from: IL-10 (e.g., pegylated IL-10, e.g., pegilodecakin 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 STAT3 downstream of IL-10R (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 (e.g., human IL-10) is present in the culture 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, the IL-10 (e.g., human IL-10) is present in the culture 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).

[0140] STAT3 activators and STAT3

[0141] In some embodiments, the IL-6R activator is a STAT3 activator. "STAT3 activator" as described herein refers to a molecule that activates STAT3 signal transduction (eg, STAT3 nuclear localization and transcription factor activity).

[0142] STAT3 is a transcription factor that exists in the cytoplasm in its inactive, non-phosphorylated form and translocates to the nucleus after it is activated by phosphorylation (e.g., Tyr705 phosphorylation) and subsequent dimerization. After entering the nucleus, the activated STAT3 dimer binds to the IFN-γ activation sequence (GAS) in the target promoter, thereby activating the transcription of the target gene. A variety of tyrosine kinases have been described as intracellular activators of STAT3 activity (e.g., JAK1, JAK2, EGFR, Src, and ERK). Additional activation mechanisms include: i) phosphorylation of STAT3 at Ser727 by protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and CDK5; and ii) acetylation of STAT3 on Lys685 by histone acetyltransferases, which can enhance STAT3 dimer stability. See, e.g., Rébé et al., JAKSTAT 2013; 2(1): e23010.

[0143] STAT3 is expressed under certain conditions in most cell types and is generally described as being associated with 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 as promoting the differentiation of macrophages toward an M2 phenotype and lacking functional dendritic cells (see, e.g., Rébé et al., 2013). STAT3 has also been described as promoting the maintenance of the pluripotent state of gp130-mediated proliferating embryonic stem cells and the macrophage differentiation of gp130-induced M1 cells. Both c-myc and pim have been identified as target genes for STAT3, and together can compensate for STAT3 in cell survival and cell cycle transformation (see, e.g., Hirano et al., Oncogene 2000; 19: 2548-2556).

[0144] STAT3 activation is rapid and transient under normal biological conditions and is mediated by many extracellular stimuli, including cytokines (IL-6, IL-10, IFNs, TNFα, LIF, OSM, etc.) and growth factors (e.g., EGF, G-CSF, GM-CSF, VEGF, HGF, GH, and Her2 / Neu). Active oncoproteins, such as Src (e.g., v-Src) and Ras, as well as chemical carcinogens and other molecules, can also activate STAT3. Indeed, many regulatory genes induced by STAT3 activity then activate the same STAT3 pathway, thereby maintaining a stable feed-forward loop. In some embodiments, the STAT3 activator includes 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 includes 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 includes a JAK activator, such as an enzyme that phosphorylates JAK (e.g., JAK2). In some embodiments, the STAT3 activator includes a hormone (e.g., leptin). In some embodiments, the STAT3 activator comprises a chaperone protein (eg, HSP90, HSP70, HSP27, HSP110, HOP).

[0145] STAT3 activity can be positively regulated by the signal transduction pathways 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 affiliated family members (e.g., IL-23) can activate STAT3 activity at least in part by promoting IL-10 / IL-10R production and autocrine signal transduction 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: 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, any one of which is described herein or known in the art.

[0146] In some embodiments, the IL-10R activator is an activator of STAT3 downstream of IL-10R. STAT3 activators may include, but are not limited to, any of the following: small molecules, nucleic acids (e.g., siRNA, shRNA, antisense RNA, microRNA), nucleic acid-based 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 targeting STAT3 phosphorylation and / or preventing STAT3 dephosphorylation), protein stabilizers (e.g., STAT3 stabilizers such as chaperones, e.g., HSP90, HSP70, HSP27, HSP110 and / or HOP), protein degradation or destabilization agents (e.g., phosphatase degradation or destabilization agents, such as PROTACs, LYTACs, molecular glues, AbTACs, CMATACs, etc., targeting phosphatases), proteins modified with unnatural amino acids, viral agents (e.g., Kaposi's sarcoma herpes virus (KSHV)), derivatives thereof, and any combination thereof.

[0147] In some embodiments, the STAT3 activator includes a cancer cell STAT3 activator. These cancer cell STAT3 activators may include any one of the following: PVT1, NEAT1, FEZF1-AS1, UICC, MALAT1, XIST, miR-30d, CD, CD146, CD24, CDK7, SOX, Smad6, Smad7, TRIM24, TRIM27, TRIM59, ADAM12, USP22, BMX, AKR1C1, PRMT1, PBX1, HSP110, RanBP6, RAC1-GTP, PA28γ, E6 and FABP5.

[0148] In some embodiments, the STAT3 activator includes a small molecule selected from the group consisting of Colivirin, Colivirin TFA, Gashengwo D, Butyzamide, Eflepedocokin α, Broussonin E, derivatives thereof, and any combination thereof. In some embodiments, the STAT3 activator includes Colivirin, Colivirin TFA, and / or Gashengwo D.

[0149] In some embodiments, the STAT3 activator includes an inhibitor or antagonist of a molecule or compound that inactivates STAT3 (e.g., reduces phosphorylated STAT3 levels) or reduces total STAT3 levels (e.g., by proteasomal degradation and / or transcriptional inhibition) in a target cell (e.g., a bone marrow cell, such as a κAPC cell). 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), minecoside, Luteolin (3,4,5,7-tetrahydroxyflavone), SHP-1, SHP-2, PTP1B, PTPRM, eEF2 kinase, PKM2, curcumin, cucurbitacin, honokiol, guggulsterone, resveratrol, berbamine, flavopiridol, JAK inhibitors / inactivated JAKs (e.g., JAK2), low molecular weight DSP2, PIAS3, etc. In some embodiments, molecules or compounds that can reduce total STAT3 levels (e.g., by proteasomal degradation and / or transcriptional inhibition) include, but are not limited to: PDLIM2, COPl, calcineurin, SOCS protein, 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 target cells competitively bind to STAT3 to prevent inactivation (e.g., DDIAS) or protein degradation (e.g., chaperone proteins, such as HSP90).

[0150] 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.

[0151] Exemplary amounts of STAT3 activators can be found, for example, in Table 1.

[0152] IL-4 receptor (IL-4R) activators and IL-4

[0153] The "IL-4 receptor (IL-4R) activator" described herein refers to a molecule that activates the IL-4 receptor-mediated signal transduction pathway.

[0154] Interleukin 4 (IL-4) is a cytokine that induces initial helper T cells (Th0 cells) to differentiate into Th2 cells. After being activated by IL-4, Th2 cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 is mainly produced by mast cells, Th2 cells, eosinophils and basophils. It is closely related to IL-13 and has similar functions to it. Interleukin 4 has many biological effects, including stimulating the proliferation of activated B cells and T cells, and the differentiation of B cells into plasma cells. It is a key regulator in humoral immunity and adaptive immunity. IL-4 induces B cell class switching to IgE and upregulates MHC class II production. IL-4 reduces the production of Th1 cells, macrophages, IFNγ and dendritic cells IL-12. IL-4 signal transduction determines the content of CD20 on the surface of normal and malignant B lymphocytes by activating the transcription factor STAT6. Excessive production of IL-4 is associated with allergies.

[0155] The IL-4 mentioned herein includes any construct having an IL-4 component (e.g., naturally produced IL-4, e.g., recombinant IL-4). These include, but are not limited to, natural IL-4 (e.g., various subtypes of human IL-4), synthetic or recombinant IL-4, and fusion proteins having an IL-4 component.

[0156] In some embodiments, the IL-4R activator further comprises a moiety that enhances stability or half-life, including, for example, an Fc moiety or a PEG moiety.

[0157] In some embodiments, the multiple S / D / M factors include an IL-4R activator, optionally wherein the IL-4R activator is selected from: 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 (e.g., human IL-4) is present in the culture 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, the IL-4 (e.g., human IL-4) is present in the culture 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).

[0158] In some embodiments, the IL-4R activator is IL-13 (such as human IL-13, or human recombinant IL-13). In some embodiments, the IL-13 is present in the culture medium at a concentration of at least about 30 pg / ml, optionally at least about 60 pg / ml, further optionally about 60 pg / ml to about 2 ng / ml (e.g., about 100 pg / ml to about 2 ng / ml).

[0159] TNFα receptor (TNFR) activators and TNFα

[0160] The "TNFα receptor (TNFR) activator" as used herein refers to a molecule that activates the TNFR-mediated signal transduction pathway. The TNFR as used herein refers to TNFR1 or TNFR2.

[0161] Tumor necrosis factor alpha (TNF, cachexin, or cachectin; commonly referred to as 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 a uniform TNFα domain.

[0162] TNFα binds 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 the membrane-bound and soluble trimeric forms of TNF, whereas TNFR2 is typically found in cells of the immune system and responds to the membrane-bound form of the TNFα homotrimer.

[0163] TNFα is 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, cardiomyocytes, 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, which is released following inflammatory stimuli (e.g., LPS).

[0164] TNFα mentioned herein includes any construct with TNFα component (e.g., naturally produced TNFα, e.g., recombinant TNFα). These include, but are not limited to: natural TNFα (e.g., various subtypes of human TNFα), synthetic or recombinant TNFα, and fusion proteins with TNFα component.

[0165] 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.

[0166] In some embodiments, the plurality of S / D / M factors include a TNFR activator, optionally wherein 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 culture 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 culture 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).

[0167] IFNγ receptor (IFNGR) activators and IFNγ

[0168] The "IFNγ receptor (INFGR) activator" described herein refers to a molecule that activates the INFGR-mediated signal transduction pathway.

[0169] Interferon gamma (IFN-γ) is a dimerized soluble cytokine, which is the only member of the type II class of interferon. IFN-γ or type II interferon is a key cytokine for innate and adaptive immunity against viruses, some bacteria and protozoa infections. IFN-γ is an important activator of macrophages and an inducer of expression of major tissue compatibility complex II molecules. Abnormal IFN-γ expression is associated with many autoinflammatory and autoimmune diseases. The importance of IFN-γ in the immune system is partly derived from its ability to directly inhibit viral replication, and most importantly from its immunostimulatory and immunomodulatory effects. IFN-γ is mainly produced by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and once antigen-specific immunity develops into part of the adaptive immune response, it is produced by CD4 Th1 and CD8 cytotoxic T lymphocytes (CTL) effector T cells. IFN-γ is also produced by non-cytotoxic innate lymphoid cells (ILC), an immune cell family first discovered in the early 2010s.

[0170] IFN-γ mentioned herein includes any construct with IFN-γ component (e.g., naturally produced IFN-γ, e.g., recombinant IFN-γ). These include, but are not limited to: natural IFN-γ (e.g., various subtypes of human IFN-γ), synthetic or recombinant IFN-γ and fusion proteins with IFN-γ component.

[0171] In some embodiments, the IFNGR activator further comprises a moiety that enhances stability or half-life, including, for example, an Fc moiety or a PEG moiety.

[0172] In some embodiments, the multiple S / D / M factors include IFNGR activators, optionally wherein the IFNGR activator is selected from: 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 (e.g., human IFNγ) is present in a culture medium at a concentration of at least about 1ng / ml, optionally at least about 5ng / ml (e.g., at least about 10ng / ml, about 20ng / ml or about 50ng / ml). In some embodiments, the IFNγ (e.g., human IFNγ) is present in a culture medium at a concentration of about 1ng / ml to about 500ng / ml (e.g., about 5ng / ml to about 200ng / ml, for example, about 10ng / ml to about 100ng / ml, for example, about 40ng / ml to about 60ng / ml, for example, about 50ng / ml).

[0173] In some embodiments, the plurality of S / D / M factors comprises two or more agents as described herein selected from the group consisting of an IL-4R activator, a TNFR activator, and an IFNGR activator.

[0174] In some embodiments, the plurality of S / D / M factors includes IL-10, IL-4, TNFα, and IFNγ.

[0175] GM-CSF receptor (GM-CSFR) activators and GM-CSF

[0176] The "GM-CSF receptor (GM-CSFR) activator" described herein refers to a molecule that activates the GM-CSFR-mediated signal transduction pathway.

[0177] 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, which functions as a cytokine. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils and basophils) and monocytes. Monocytes exit the circulation and migrate into tissues, where they then mature into macrophages and dendritic cells. It is part of the immune / inflammatory cascade, and the activation of a small number of macrophages can rapidly lead to an increase in their number, which is a key process in fighting infection. GM-CSF also has some 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.

[0178] GM-CSF mentioned herein includes any construct having a GM-CSF component (e.g., naturally produced GM-CSF, e.g., recombinant GM-CSF). These include, but are not limited to, natural GM-CSF (e.g., various isoforms of human GM-CSF), synthetic or recombinant GM-CSF, and fusion proteins having a GM-CSF component.

[0179] In some embodiments, the GM-CSFR activator further comprises a moiety that enhances stability or half-life, including, for example, an Fc moiety or a PEG moiety.

[0180] In some embodiments, the multiple S / D / M factors also include a GM-CSF receptor (GM-CSFR) activator. In some embodiments, the GM-CSFR activator is selected from: 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 (e.g., human GM-CSF) is present in the culture medium at a concentration of at least about 30pg / ml, optionally at least about 50pg / ml (e.g., at least about 100pg / ml, about 150pg / ml, about 200pg / ml or about 300pg / ml). In some embodiments, the GM-CSF (e.g., human GM-CSF) is present in the culture 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).

[0181] IL-6 receptor (IL-6R) activators and IL-6

[0182] The "IL-6 receptor (IL-6R) activator" described herein refers to a molecule that activates the IL-6 receptor-mediated signal transduction pathway.

[0183] 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 that respond to specific microbial molecules, known as pathogen-associated molecular patterns (PAMPs). These PAMPs bind to an important group of detection molecules of the innate immune system, known as pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and intracellular compartments, and induce intracellular signal cascades to cause inflammatory cytokine production. IL-6 is an important mediator of fever and acute phase reactions. IL-6 is responsible for stimulating acute phase protein synthesis, and the production of neutrophils in the bone marrow. It supports B cell growth and antagonizes regulatory T cells.

[0184] IL-6 mentioned herein includes any construct having an IL-6 component (e.g., naturally produced IL-6, e.g., recombinant IL-6). These include, but are not limited to: natural IL-6 (e.g., various subtypes of human IL-6), synthetic or recombinant IL-6, and fusion proteins having an IL-6 component.

[0185] In some embodiments, the IL-6R activator further comprises a moiety that enhances stability or half-life, including, for example, an Fc moiety or a PEG moiety.

[0186] In some embodiments, the multiple S / D / M factors also include an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from: 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 human IL-6, or human recombinant IL-6. In some embodiments, the IL-6 (e.g., human IL-6) is present in the culture 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, the IL-6 (e.g., human IL-6) is present in the culture 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).

[0187] In some embodiments, the plurality of S / D / M factors includes IL-10, IL-4, TNFα, IFNγ, GM-CSF, and IL-6.

[0188] In some embodiments, multiple S / D / M factors described herein are present in a single composition.

[0189] In some embodiments, the plurality of S / D / M factors described herein further comprises one or more cytokines selected from the group consisting of IL-2, IL-17 (eg, IL-17A), and / or M-CSF.

[0190] In some embodiments, at least one of the plurality of S / D / M factors (eg, IL-10) is provided separately from the other S / D / M factors in the plurality of S / D / M factors.

[0191] Optimization Factor

[0192] In some embodiments, the method further comprises: after contacting the plurality of monocytes with a plurality of S / D / M factors or a culture medium derived from a T cell culture, contacting the plurality of monocyte populations with a plurality of optimization factors. The optimization factors are selected from: type I interferons (such as IFNα and / or IFNβ), IFNγ, TNFα, TLR ligands (such as poly IC, CpG or LPS), CD40L or CD40-ligated antibodies, anti-PD-L1 antibodies and TPI-1.

[0193] In some embodiments, a method of optimizing a population of APCs is provided, comprising contacting the population of APCs with a) IFNα, b) IFNγ, and c) TNFα.

[0194] In some embodiments, a method of optimizing a population of APCs is provided, comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) TNFα, d) poly IC, and e) CpG.

[0195] In some embodiments, a method of optimizing an APC population is provided, comprising contacting the APC population with a) IFNα, b) IFNγ, c) TNFα, d) poly IC, e) CD40L, and f) an anti-PD-L1 antibody.

[0196] In some embodiments, a method of optimizing an APC population is provided, comprising contacting the APC population with a) IFNα, b) IFNγ, c) TNFα, d) poly IC, e) CD40L, f) anti-PD-L1 antibody, and g) a SHP-1 inhibitor (e.g., TPI-1).

[0197] In some embodiments, a method of optimizing a population of APCs is provided, comprising contacting the population of APCs with a) IFNα, b) IFNγ, c) R848, d) poly IC, and e) a SHP-1 inhibitor (eg, TPI-1).

[0198] In some embodiments, a method of optimizing a population of APCs is provided, comprising contacting the population of APCs with a) IFNγ, b) R848, c) poly IC, and d) a SHP-1 inhibitor (eg, TPI-1).

[0199] In some embodiments, a method of optimizing an APC population is provided, comprising contacting the APC population with a) IFNα, b) IFNγ, c) poly IC, d) CpG, e) CD40L, f) anti-PD-L1 antibody, g) SHP-1 inhibitor (e.g., TPI-1) and h) TNFα.

[0200] In some embodiments, multiple optimization factors are provided immediately after contacting multiple monocytes with multiple S / D / M factors or culture medium derived from T cells. In some embodiments, multiple optimization factors are provided within about 1 day after contacting multiple monocytes with multiple S / D / M factors or culture medium derived from T cells.

[0201] In some embodiments, the plurality of monocytes is cultured in the presence of a plurality of optimizing factors for about 1-5 days (eg, about 1, 2, 3, 4, or 5 days).

[0202] In some embodiments, a plurality of optimizing factors is provided when at least about 50% (e.g., about 50%, 60%, 70%, 80%, or 99%) of the monocytes survive after contacting the plurality of monocytes with the plurality of S / D / M factors or culture medium derived from a culture of T cells.

[0203] In some embodiments, multiple optimization factors are provided when at least about 10%, 20%, 30%, 40%, or 50% of the monocytes display dendritic cell morphology.

[0204] In some embodiments, when the monocytes express high levels of one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86 and / or CD40, multiple optimization factors are provided. In some embodiments, when the monocytes express one or more molecules selected from the group consisting of MHC I, MHC II, CD80, CD86 and / or CD40 at higher levels (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) compared to monocytes obtained from the same individual and cultured with GM-CSF and M-CSF (e.g., at concentrations routinely used in the art of these methods), multiple optimization factors are provided.

[0205] In some embodiments, the various optimization factors described herein can be used to prime APCs (eg, APCs obtained from humans) independently of the above methods.

[0206] In some embodiments, the optimizing factors include IFNα, IFNγ, and TNFα.

[0207] In some embodiments, the optimization factors include IFNα, IFNγ, TNFα, poly IC, and CpG.

[0208] In some embodiments, the optimizing factors include IFNα, IFNγ, TNFα, poly IC, CpG, CD40L, and anti-PD-L1 antibodies.

[0209] In some embodiments, the optimization factors include IFNα, IFNγ, TNFα, poly IC, CpG, CD40L, anti-PD-L1 antibody and TPI-1.

[0210] In some embodiments, the concentration of IFNα in the optimizer is about 1 ng / ml to about 50 ng / ml (eg, about 5 ng / ml to about 20 ng / ml, eg, about 10 ng / ml).

[0211] In some embodiments, the concentration of IFNγ in the optimizer 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).

[0212] In some embodiments, the concentration of TNFα in the optimizer is about 1 ng / ml to about 50 ng / ml (eg, about 5 ng / ml to about 20 ng / ml, eg, about 10 ng / ml).

[0213] In some embodiments, the concentration of poly IC in the optimizer 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).

[0214] In some embodiments, the concentration of CpG in the optimizer 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).

[0215] In some embodiments, the concentration of CD40L in the optimization 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).

[0216] In some embodiments, the concentration of the anti-PD-L1 antibody in the optimizer is about 1 μg / ml to about 200 μg / ml (e.g., about 5 μg / ml to about 100 μg / ml, e.g., about 10 μg / ml to about 50 μg / ml, e.g., about 20 μg / ml).

[0217] In some embodiments, the concentration of TPI-1 in the optimizer 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).

[0218] In some embodiments, the concentration of R848 in the optimizer 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).

[0219] Methods for promoting the survival of monocytes

[0220] The application provides various methods for promoting the survival of monocytes. In some embodiments, the monocytes are obtained (e.g., newly separated) from an individual (e.g., a human). In some embodiments, the individual suffers from cancer (e.g., any type or kind of cancer described herein). In some embodiments, the individual suffers from a disease or condition associated with immunosuppression (e.g., fibrosis after organ transplantation under immunosuppressive drugs). In some embodiments, the individual has a viral infection. In some embodiments, compared with those monocytes obtained from a reference individual (e.g., a healthy individual), the monocytes obtained from the individual express lower levels of IL-10 receptors ("IL-10R"), IL-4 receptors ("IL-4R"), IL-6 receptors ("IL-6R"), M-CSF receptors ("GM-CSFR") and / or M-CSF receptors ("GM-CSFR").

[0221] In some embodiments, the present application provides a method for promoting the survival of a monocyte population derived from an individual in an 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: IL-10 (e.g., pegylated IL-10, e.g., pegylated iloleukin 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 STAT3 downstream of IL-10R (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, the IL-10 is present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the monocyte population prior to contact with an IL-10R activator expresses lower levels of IL-10R (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) 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: TNFα, TNFR agonist antibodies, and small molecule activators of TNFR, and optionally wherein the IFNGR activator is selected from: IFNγ, IFNGR agonist antibodies, and small molecule activators of IFNGR, further optionally the culture comprises TNFα and / or IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the TFNα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml). In some embodiments, the individual has cancer (eg, a solid tumor).

[0222] In some embodiments, the present application provides a method for promoting the survival of a monocyte population from an individual in an in vitro culture, comprising culturing the monocyte population in a culture medium with a TNFα receptor (TNFR) activator and / or an interferon γ (IFNγ) receptor (IFNGR) activator, optionally wherein the TNFR activator is selected from: TNFα, TNFR agonist antibodies and small molecule activators of TNFR, and optionally wherein the IFNGR activator is selected from: IFNγ, IFNGR agonist antibodies and small molecule activators of IFNGR, further optionally the culture contains TNFα and / or IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the TFNα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml). In some embodiments, the monocyte population expresses lower levels of IL-10R (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) prior to contact with an 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).

[0223] In some embodiments, the present application provides a method for promoting the survival of a monocyte population of an individual origin in an in vitro culture, comprising culturing the monocyte population in a culture medium with 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 a culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, and further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the IFNγ is present in a culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, and further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0224] 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, 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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).

[0225] In some embodiments, the culture further comprises an IL-6 receptor (IL-6R) activator. In some embodiments, the IL-6R activator is selected from: 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 human IL-6, or human recombinant IL-6. In some embodiments, the IL-6 is present in the culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, e.g., about 30 pg / ml).

[0226] In some embodiments, the present application provides a method for promoting the survival of a monocyte population of individual origin in an in vitro culture, comprising culturing the monocyte population in a culture medium derived from a T cell after treatment with an anti-CD3 antibody and an anti-CD28 antibody, wherein the culture medium comprises an activator of IL-10R. In some embodiments, the T cell is a CD4 T cell. In some embodiments, the T cell is separated from the PBMC of the same individual or different individuals, and is not treated with anti-CD3 antibodies and / or anti-CD28 antibodies in advance before treatment. In some embodiments, the T cell is separated from the PBMC of the same individual or different individuals, and has been treated with anti-CD3 antibodies and / or anti-CD28 antibodies in advance before treatment. In some embodiments, the culture medium is derived from a culture after the T cell is treated with anti-CD3 antibodies and anti-CD28 antibodies for about 1-3 days (optionally about 2 days). In some embodiments, the population of monocytes prior to contacting with an IL-10R activator expresses lower levels of IL-10R (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) than monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the individual has cancer (e.g., a solid tumor).

[0227] The anti-CD3 / CD28 treatment of T cells described herein is a well-known technique in the art of activating T cells.

[0228] The present application also provides a method of increasing the expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual suffering from 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 lower levels of IL-10R (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) 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: IL-10 (e.g., PEGylated IL-10, e.g., PEGylated iloleukin 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 STAT3 downstream of IL-10R (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 present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 20 ng / ml). In some embodiments, the monocyte population expresses lower levels of IL-10R (at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) 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: TNFα, TNFR agonist antibodies, and small molecule activators of TNFR. In some embodiments, the IFNGR activator is selected from: IFNγ, IFNGR agonist antibodies, and small molecule activators of IFNGR. In some embodiments, the IFNGR activator includes TNFα. In some embodiments, the IFNGR activator includes IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the TFNα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml).In some embodiments, the individual has cancer (eg, a solid tumor).

[0229] In some embodiments, the monocytes are cultured for at least about 2 days (eg, about 2-3 days).

[0230] Method for promoting differentiation of monocyte population into antigen presenting cells (APC)

[0231] The present application provides a method for promoting the differentiation of a monocyte population from an individual (e.g., a cancer patient or a patient with a viral infection) into an antigen presenting cell (APC) in an in vitro culture, comprising culturing the monocyte population in a culture 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 γ (IFNγ) receptor (IFNGR) activator (e.g., IFNγ), optionally wherein the monocytes have been contacted with an IL-10 receptor (IL-10R) activator (e.g., IL-10), or the culture medium further comprises an IL-10 receptor (IL-10R) activator (e.g., IL-10). In some embodiments, a method is provided for promoting differentiation of a monocyte population derived from an individual (e.g., a cancer patient) into antigen presenting cells (APCs) in an in vitro culture, comprising culturing the monocyte population 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 γ (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, the IL-4 is present in the culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally about 30 pg / ml to about 1 ng / ml (e.g., 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, the TNFα is present in the culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml). 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml). In some embodiments, the culture medium 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, e.g., about 30 pg / ml). In some embodiments, monocytes obtained from the individual express lower levels of IL-4 receptor ("IL-4R") (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower) than those monocytes obtained from a reference individual (e.g., a healthy individual).

[0232] In some embodiments, a method is provided for promoting the differentiation of a monocyte population derived from an individual (e.g., a cancer patient) into an antigen presenting cell (APC) in an in vitro culture, comprising culturing the monocyte population in a culture medium with 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 culture medium at a concentration of at least about 15pg / ml, optionally at least about 30pg / ml, further optionally about 30pg / ml to about 1ng / ml (e.g., about 100pg / ml to about 1ng / ml). In some embodiments, the TNFα is human TNFα, or human recombinant TNFα. In some embodiments, the TNFα is present in the culture medium at a concentration of at least about 0.5ng / ml, optionally at least about 1ng / ml, further optionally about 0.5ng / ml to about 30ng / ml (e.g., about 1-10ng / ml). In some embodiments, the IFNγ is human IFNγ, or human recombinant IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally 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 levels of IL-4 receptor ("IL-4R") (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) compared to those monocytes obtained from a reference individual (e.g., a healthy individual).

[0233] In some embodiments, a method of promoting differentiation of a population of monocytes from an individual (e.g., a cancer patient) into antigen presenting cells (APCs) in an in vitro culture is provided, comprising culturing the population of monocytes in a medium having IL-4, IL-6, TNFα, and IFNγ, optionally wherein the monocytes have a lower level of IL-4 receptor ("IL-4R") expression (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally about 30 pg / ml to about 1 ng / ml (e.g., 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 culture medium at a concentration of at least about 0.5ng / ml, optionally at least about 1ng / ml, further optionally about 0.5ng / ml to about 30ng / ml (e.g., about 1-10ng / ml). In some embodiments, the IFNγ is human IFNγ, or human recombinant IFNγ. In some embodiments, the IFNγ is present in the culture medium at a concentration of at least about 5ng / ml, optionally at least about 10ng / ml, further optionally about 10ng / ml to about 200ng / ml (e.g., about 50-100ng / 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 culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, e.g., about 30 pg / ml).

[0234] 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, 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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).

[0235] In some embodiments, the culture further comprises an IL-10 receptor (IL-10R) activator. In some embodiments, the IL-10R activator is selected from: IL-10 (e.g., PEGylated IL-10, e.g., PEGylated iloleukin 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 STAT3 downstream of IL-10R (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 present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (eg, about 20 ng / ml).

[0236] In some embodiments, the monocytes are cultured for at least about 2 days (eg, about 2-3 days).

[0237] Method for obtaining tumor-specific or virus-specific antigen-presenting cells

[0238] In some embodiments, a method for obtaining tumor-specific antigen-presenting cells is provided, comprising: a) contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors (S / D / M factors), respectively or simultaneously, wherein the plurality of S / D / M factors comprise: 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 γ (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 tumor-specific APCs.

[0239] In some embodiments, the composition comprising a tumor antigen includes tumor cells (e.g., a tumor biopsy or cells from a patient, e.g., a fresh or frozen-thawed tumor biopsy sample, e.g., cultured tumor cells from a tumor biopsy).

[0240] In some embodiments, the composition comprising a tumor antigen includes a neoantigenic peptide (e.g., a personalized neoantigenic long peptide (LP)). In some embodiments, the neoantigenic peptide is recognized and synthesized by an AI. See, e.g., Mor SK et al., Oncoimmunology. 2022 Jan 10; 11(1):2023255.

[0241] In some embodiments, the composition comprising a tumor antigen includes a shared tumor-associated antigen. In some embodiments, the shared tumor-associated antigen includes an autologous antigen (e.g., an abnormally expressed autologous antigen). In some embodiments, the autologous antigen is selected from: melanoma antigen-1 (MAGE-1), prostate-related PAP, PSA and PSMA, breast cancer-related BCAR3 and multiple cancer-related MUC1. In some embodiments, the shared tumor-associated antigen includes a non-self antigen of viral origin (e.g., an antigen from LMP1 / 2 associated with nasopharyngeal carcinoma and lymphoma, for example, an antigen from the E6 and E7 proteins of high-risk human papillomavirus (HPV), for example, an antigen from the retrovirus Tax protein found in adult T-cell leukemia). In some embodiments, the shared tumor-associated antigen includes a new antigen caused by a mutation shared in different types of cancer (e.g., a new antigen associated with a p53 mutation or a KRAS mutation).

[0242] In some embodiments, the tumor antigen peptide (e.g., a synthetic tumor antigen peptide) is obtained by: a) determining a tumor-specific mutation in a tumor tissue sample of a patient with a virus-related cancer, wherein the tumor-specific mutation is not present in the virus, and b) synthesizing a peptide comprising the tumor-specific mutation. In some embodiments, the tumor-specific mutation is determined by sequencing the tumor tissue sample and the virus sample and comparing the sequences from the two samples.

[0243] In some embodiments, the tumor antigen peptide (e.g., a synthetic tumor antigen peptide) is obtained by: a) determining a tumor-specific mutation in a tumor tissue sample of a patient with cancer, the tumor-specific mutation not being present in a normal tissue sample of the cancer patient, and b) synthesizing a peptide comprising the tumor-specific mutation. In some embodiments, the tumor-specific mutation is determined by sequencing the tumor tissue sample and the normal tissue sample and comparing the sequences from the two samples.

[0244] In some embodiments, a method for obtaining tumor-specific antigen presenting cells is provided, comprising: a) contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 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; and b) introducing a polynucleotide encoding a tumor antigen (e.g., any tumor antigen described herein) into the population of APCs. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is mRNA.

[0245] In some embodiments, a method for obtaining virus-specific antigen presenting cells is provided, comprising: a) contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 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; and b) contacting a composition comprising a viral antigen with the population of APCs, thereby obtaining virus-specific APCs. In some embodiments, the composition comprising a viral antigen comprises a viral sample.

[0246] In some embodiments, a method for obtaining virus-specific antigen presenting cells is provided, comprising: a) contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 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; 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.

[0247] In some embodiments, a tumor-specific APC population or a virus-specific APC population produced by any of the methods described herein is provided.

[0248] Monocytes

[0249] The methods described herein convert a plurality of monocytes into APCs. In some embodiments, the plurality of monocytes are obtained from the peripheral blood of an individual.

[0250] In some embodiments, when monocytes are obtained from peripheral blood, the monocytes express CD14. The method of obtaining monocytes from peripheral blood is well known in the art. For example, PBMCs can be planted on a cell culture dish to allow monocytes to adhere, which is a common method for separating them from non-adherent lymphocytes. It is also possible to separate monocytes by positive selection using anti-CD14 antibodies, or by negative selection using all antibodies for other cells.

[0251] In some embodiments, monocytes obtained from an individual express lower levels of IL-10 receptor ("IL-10R") prior to contact with one or more of the above cytokines, compared to those monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the amount of IL-10R on monocytes from an individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than the amount of IL-10R on monocytes from a reference individual (e.g., a healthy individual).

[0252] In some embodiments, monocytes obtained from an individual express lower levels of IL-4 receptor ("IL-4R") prior to contact with one or more of the above cytokines, compared to those monocytes obtained from a healthy individual. In some embodiments, the amount of IL-4R on monocytes from an individual is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% lower than the amount of IL-4R on monocytes from a reference individual (e.g., a healthy individual).

[0253] In some embodiments, monocytes obtained from an individual express lower levels of IL-6 receptor ("IL-6R") prior to contact with one or more of the above-mentioned cytokines, compared to those monocytes obtained from a healthy individual. In some embodiments, the amount 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 amount of IL-6R on monocytes from a reference individual (e.g., a healthy individual).

[0254] In some embodiments, monocytes obtained from an individual prior to contact with one or more cytokines described above express lower levels of M-CSF receptor ("M-CSFR") compared to those monocytes obtained from a healthy individual. In some embodiments, the amount 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 amount of M-CSFR on monocytes from a reference individual (e.g., a healthy individual).

[0255] In some embodiments, monocytes obtained from an individual prior to contact with one or more of the above-described cytokines express lower levels of GM-CSF receptor ("GM-CSFR") than those monocytes obtained from a healthy individual. In some embodiments, the amount 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 amount of GM-CSFR on monocytes from a reference individual (e.g., a healthy individual).

[0256] In some embodiments, the methods described herein further comprise assessing the monocytes' expression level of IL-10R (eg, prior to contacting the monocytes with one or more cytokines (eg, IL-10, IFNγ, and / or TNFα)).

[0257] cancer

[0258] The cancer described in this section (eg, in the context of monocytes derived from cancer patients) can be of any type or kind. All cancer types discussed in Section V apply similarly here.

[0259] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood cancer.

[0260] 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 malignancy.

[0261] Examples of cancers described herein include, but are not limited to, adrenocortical carcinoma, myeloid metaplasia of unknown etiology, 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 tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., fibrillary astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic gliomas and glioblastomas), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary, 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 tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., liver cancer and hepatoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), 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, and lung squamous cell carcinoma), lymphoid tumors (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma, lymphoma Examples include leukemia, primary central nervous system lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, colorectal 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, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor and post-transplantation lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0262] In some embodiments, the cancer is a viral infection-related cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-related cancer (e.g., HPV-related cervical cancer, e.g., HPV-related head and neck cancer, e.g., HPV-related squamous cell carcinoma). In some embodiments, the cancer is a human herpesvirus 8 (HHV8)-related cancer (e.g., Kaposi's sarcoma). In some embodiments, the cancer is a human T-lymphotrophic virus (HTLV-1)-related cancer (e.g., adult T-cell leukemia or lymphoma). In some embodiments, the cancer is an Epstein-Barr virus (EBV)-related cancer (e.g., Burkitt's lymphoma, Hodgkin's and non-Hodgkin's lymphoma, gastric cancer). In some embodiments, the cancer is a hepatitis B virus (HBV)-related cancer (e.g., liver cancer). In some embodiments, the cancer is a hepatitis C virus-related cancer (e.g., liver cancer, non-Hodgkin's lymphoma).

[0263] In some embodiments, the cancer is refractory to one or more of radiation therapy, chemotherapy, or immunotherapy (eg, checkpoint blockade).

[0264] In some embodiments, the cancer is liver cancer, kidney cancer, endometrial cancer, thymic epithelial tumor, lung cancer, spindle cell sarcoma, chondrosarcoma, uterine smooth muscle, pancreatic cancer.

[0265] individual

[0266] In some embodiments, the individual is healthy or has no symptoms of a disease or condition (e.g., cancer). In some embodiments, the individual has a cancer or tumor. In some embodiments, the individual has a solid tumor. In some embodiments, the cancer is a blood cancer.

[0267] In some embodiments, the individual has advanced cancer. In some embodiments, the individual has late stage cancer. In some embodiments, the individual has cancer in stage II, III, or IV. In some embodiments, the individual has an inoperable tumor and / or metastasis. In some embodiments, the cancer is malignant.

[0268] In some embodiments, the individual has an infection (eg, a viral infection, a bacterial infection).

[0269] In some embodiments, the individual is female. In some embodiments, the individual is male.

[0270] 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 old.

[0271] III. Antigen Presenting Cells (APCs), Compositions and Cultures.

[0272] The present application provides APCs, such as those prepared according to any of the above methods, having unique properties that distinguish them from naturally occurring APCs or APCs produced in vitro by currently known methods. Comprehensive studies discussed in detail in the Examples show that the exemplary APCs of the present application are different from (e.g.) dendritic cells (e.g., cDC1, cDC2, pDC) or macrophages (e.g., M1 macrophages, M2 macrophages) in terms of shape / size, how they adhere to the matrix substrate, various cell surface molecules, antigen presentation ability and / or gene transcription profile. See, for example, Figures 14 to 19C. The results overall show that the exemplary APCs of the present application are a unique cell population that is generally smaller (about 7-15 μm after trypsinization) than macrophages / dendritic cells (about 10 to 20 μm) that have different shapes, superior antigen presentation ability, and are highly sensitive to various types of antigens, as confirmed by high expression of, for example, CD40, TLR2 and STING. Exemplary APCs uniquely express high levels of LOX1 and uPAR and have a very different gene transcription profile compared to mature DCs or macrophages. These data demonstrate that the APCs of the present application represent a novel and powerful APC population that is fundamentally different from currently known APCs.

[0273] The exemplary APCs described herein have surface markers different from known dendritic cell subsets (such as myeloid cDC1, myeloid cDC2, plasmacytoid DC (pDC) or Mo-DC) and known human macrophages (such as M1 macrophages and M2 macrophages) or monocytes. See, for example, Figures 18A to 18B, 19A to 19C and 15A to 15B. For example, while cDC1 expresses high levels of CD141, CLEC9A, XCR1, CD103, CD103 and DEC205, 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 than both of them. pDC expresses high levels of CD303, while the exemplary APCs of the present application express much lower levels than CD303. MoDC expresses high levels of DEC205, SIRPa, CD1c, CD11c, CD303 and CD209, while the exemplary APCs of the present application do not express or express much lower levels of any of those surface markers. M1 macrophages express high levels of CD26, SIRPa, CD11c, CCR2, CCR7, CD14, CD303, PD-L1, CLEC5, and CCR1, while the exemplary APCs of the present application have similar expression of CD14 and PD-L1, but different expression of all other surface markers.

[0274] On the other hand, the exemplary APCs of the present application consistently express several surface molecules that are not expressed or expressed at relatively low levels in the above-mentioned dendritic cells or macrophages. These molecules include the receptor for oxidized LDL (LOX1), urokinase-type plasminogen activator receptor (uPAR), IL-3 receptor (IL-3R) and the receptor for complement component 3a (C3AR), TLR2 and / or STING. None of the dendritic cells or macrophages have a similar expression pattern of these molecules.

[0275] Consistently, the exemplary APCs of the present application have different morphologies, as shown, for example, in FIG. 14. These APCs also exhibit different gene transcription profiles, such as Figures 16A to 16C As shown in . As shown, these cells are smaller than dendritic cells or macrophages and have different cell shapes. Although these exemplary APCs have unique patterns of gene transcription profiles with dendritic cells, macrophages or monocytes, a) monocytes derived from cancer patients, b) monocytes derived from healthy individuals, c) cultured with KX1, and d) these exemplary APCs cultured with c-combo, show highly similar gene transcription profiles. These results once again confirm that the APCs of the present application are unique and non-transient APC populations.

[0276] In some embodiments, a population of APCs is provided, wherein the APCs 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) co-stimulatory 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 lower levels of 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, e) are cytosolic and cytosolic with cDC1, In some embodiments, the APCs express at least one cDC2 surface molecule including SIRPα and CD1c compared to C2, f) express at least one or more (e.g., 2, 3, 4, or 5) dendritic cell or macrophage surface molecules including CD11c, CCR2, CCR7, CD14, and CD303 compared to MoDC (e.g., those derived from monocytes after LPS or TNFα treatment), 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-MoDC, 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., monocytes derived therefrom). In some embodiments, the APC is CD31+ / low, or has reduced CD31 compared to monocytes (e.g., monocytes derived therefrom). In some embodiments, the APC is PD-L1+ / high, or has increased PD-L1 compared to monocytes (e.g., monocytes derived therefrom). In some embodiments, the APC is SIRPa- / low, LilRB- / low and / or Siglec- / low, or has reduced SIRPa, LilRB and Siglec expression compared to monocytes (e.g., monocytes derived therefrom). In some embodiments, the APC is CD32+ / high, Trem2 high, IL-3R high and / or c-Met+ / high. In some embodiments, the APC is TLR3+ / high, and / or TLR8+ / high (e.g., having higher TLR3 and / or TLR8 expression than M1 macrophages). In some embodiments, the APC expresses lower levels of CD14 compared to monocytes. In some embodiments, the APC has a size less than about 15 μm (diameter) (eg, about 7-15 μm).In some embodiments, when the APCs adhere to the matrix, they have multiple shapes (polymorphic) and can elongate / stretch to adopt a spindle shape. After trypsinization, the cells aggregate together and are uniform in size, less than about 15 μm (e.g., about 7-15 μm).

[0277] As described herein, "+ / high" refers to the positive expression of a surface molecule, or the high expression of a surface molecule. In some embodiments, high expression refers to a scenario in which the cell population (e.g., APCs) 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) known to express this surface molecule. In some embodiments, a higher level refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, or 100 times higher than the expression level. In some embodiments, the expression of a certain molecule that is increased refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times or 100 times higher than the expression level. In some embodiments, the expression of a certain molecule that is reduced refers to an expression level that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% lower.

[0278] 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 at least 5-fold or 10-fold higher CD40 surface expression levels compared to dendritic cells (e.g., dendritic cells derived from monocytes treated with GM-CSF / IL-4 followed by LPS, as described herein) or M1 / M2 macrophages (e.g., M1 / M2 macrophages derived from monocytes treated with M-CSF followed by LPS and IFNγ for an M1 phenotype, or M1 / M2 macrophages derived from monocytes treated with LPS, IL4, and IL-10 for an M2 phenotype). In some embodiments, the APC has comparable or higher MHC-I, MHC-II, CD80, CD86, and CD40 surface expression compared to dendritic cells (e.g., dendritic cells derived from monocytes treated with GM-CSF / IL-4 followed by LPS, as described herein) or M1 / M2 macrophages (e.g., for the M1 phenotype, monocytes derived from M-CSF followed by LPS and IFNγ, or for the M2 phenotype, M1 / M2 macrophages derived from monocytes treated with LPS, IL4, and IL-10). In some embodiments, the APC is IL-3R+ / high, TREM2+ / high, C3AR+ / high, and PD-L1+ / high. In some embodiments, the size of the APC is less than about 15 μm (diameter) (e.g., about 7-15 μm). In some embodiments, when the APCs are attached to a matrix, they have a variety of shapes (multi-shape) and can be elongated / stretched to adopt a spindle shape. After trypsinization, the cells aggregated together and were uniform in size, less than about 15 μm (eg, about 7-15 μm).

[0279] In some embodiments, a population of APCs is provided, such as APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), wherein the APCs a) are MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II than 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, when the APCs are attached to a matrix, they have a variety of shapes (polymorphic) and can stretch / elongate to take a spindle shape. After trypsinization, these cells aggregate 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, it is TLR2+ / high and STING+ / high (e.g., expressing higher levels of TLR2 and STING than M1 macrophages), and / or expresses higher levels of CD40 compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs. In some embodiments, the APC a) does not express or expresses lower levels of CD141, XCR1, and CD103 compared to cDC1, b) does not express or expresses lower levels of CD1c and SIRPα compared to cDC2, c) expresses lower levels of CD303 compared to pDC or M1 macrophages, d) expresses lower levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, the APC has a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APC has a fusiform or elongated shape or multiple shapes. In some embodiments, the APCs are attached to the matrix substrate but can be easily removed by repeated pipetting or brief trypsinization (<2 min at 37°C). In some embodiments, the APCs a) are CD80+ / high and CD86+ / high (e.g., express higher levels of CD80 and CD86 than monocytes, M1 macrophages, M2 macrophages, and MoDCs). In some embodiments, the CD40 expression level on the APCs is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level of CD40 on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0280] In some embodiments, a population of APCs is provided (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), wherein the APCs 1) are MHC-I+ / high and MHC-II+ / high (e.g., expressing higher levels of MHC-I and MHC-II than monocytes, M1 macrophages, M2 macrophages, and MoDCs), and 2) a) do not express or express lower levels of CD141, XCR1, and CD103 than cDC1, b) do not express or express lower levels of CD1c and SIRPα than cDC2, c) express lower levels of CD303 than pDC or M1 macrophages, and / or d) express lower levels of CCR7, CCR2, and CD11c than 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 APC a) is CD80+ / high and CD86+ / high (e.g., expressing higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDC). In some embodiments, the APC expresses comparable or higher levels of CD80, CD86, CD40, OX40L, ICOSL, and / or CD70 compared to mature dendritic cells (e.g., monocytes derived from cultured with LPS or TNFα). In some embodiments, the APC expresses higher levels of LOX1, uPAR, CD40, TLR2, IL-3R, TREM2, C3AR, IL-3R, and / or PD-L1 compared to mature dendritic cells (e.g., monocytes derived from cultured with LPS or TNFα), monocytes, M1 or M2 macrophages.

[0281] In some embodiments, a population of APCs is provided, such as APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), wherein the APCs a) are MHC-I+ / high and MHC-II+ / high (e.g., express higher levels of MHC-I and MHC-II than 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 than monocytes, M1 macrophages, M2 macrophages, and MoDCs. In some embodiments, the APC a) does not express or expresses lower levels of CD141, XCR1, and CD103 as compared to cDC1, b) does not express or expresses lower levels of CD1c and SIRPα as compared to cDC2, c) expresses lower levels of CD303 as compared to pDC or M1 macrophages, d) expresses lower levels of CCR7, CCR2, and CD11c as compared to M1 macrophages. In some embodiments, the APC has a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APC has a fusiform or elongated shape or multiple shapes. In some embodiments, the APC is attached to the matrix substrate, but can be easily removed by repeated pipetting or brief trypsinization (<2 min at 37°C). In some embodiments, the APC a) is CD80+ / high and CD86+ / high (e.g., expressing higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDC). In some embodiments, the expression level of CD40 on the APC is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level of CD40 on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0282] In some embodiments, a population of APCs is provided (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), wherein the APCs a) are 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), b) express or express high levels of at least one of LOX1 and uPAR (e.g., expressing higher levels of LOX1 and uPAR compared to monocytes, M1 macrophages, M2 macrophages, and MoDCs), and c) express higher 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., expressing higher levels of TLR2 and STING than M1 macrophages). In some embodiments, the APC a) does not express or expresses lower levels of CD141, XCR1, and CD103 compared to cDC1, b) does not express or expresses lower levels of CD1c and SIRPα compared to cDC2, c) expresses lower levels of CD303 compared to pDC or M1 macrophages, d) expresses lower levels of CCR7, CCR2, and CD11c compared to M1 macrophages. In some embodiments, the APC has a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APC has a fusiform or elongated shape or multiple shapes. In some embodiments, the APC is attached to the matrix substrate, but can be easily removed by repeated pipetting or brief trypsinization (<2 min at 37°C). In some embodiments, the APC a) is CD80+ / high and CD86+ / high (e.g., expressing higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages, and MoDC). In some embodiments, the expression level of CD40 on the APC is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level of CD40 on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0283] In some embodiments, a population of APCs is provided (e.g., APCs derived from monocytes obtained from an individual (e.g., a healthy individual, a cancer patient, or a virally infected patient), wherein 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., express 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, the APC a) does not express or expresses lower levels of CD141, XCR1 and CD103 compared to cDC1, b) does not express or expresses lower levels of CD1c and SIRPα compared to cDC2, c) expresses lower levels of CD303 compared to pDC or M1 macrophages, d) expresses lower levels of CCR7, CCR2 and CD11c compared to M1 macrophages. In some embodiments, the APC has a size of less than about 15 μm (e.g., about 7-15 μm). In some embodiments, the APC has a fusiform or elongated shape or multiple shapes. In some embodiments, the APC is attached to the matrix substrate, but can be easily removed by repeated pipetting or brief trypsinization (<2 min at 37°C). In some embodiments, the APC a) is CD80+ / high and CD86+ / high (e.g., expressing higher levels of CD80 and CD86 compared to monocytes, M1 macrophages, M2 macrophages and MoDC). In some embodiments, the expression level of CD40 on the APC is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than the expression level of CD40 on monocytes, M1 macrophages, M2 macrophages, and MoDCs.

[0284] 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, wherein the APCs a) have a spindle or elongated shape or multiple shapes, b) express higher or comparable levels of MHC-I, MHC-II, CD80, CD86, CD40, OX40L, ICOSL and / or CD70 compared to mature dendritic cells (e.g., monocytes derived from monocytes cultured with LPS or TNFα), c) 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., monocytes derived from monocytes cultured with LPS or TNFα), monocytes, M1 or M2 macrophages, d) express lower levels of DEC205, SIRPα and / or CD11c compared to mature dendritic cells (e.g., derived from monocytes after culture with LPS or TNFα) or M1 macrophages, e) express lower 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 compared to M1 macrophages, and / or g) express higher levels of Sting and / or TLR2, optionally TLR3 and / or TLR8 compared to M1 macrophages.

[0285] In some embodiments, compared with mature dendritic cells (e.g., derived from monocytes after culture with LPS), the APC expresses higher levels of CCL3L1, CXCL8, IL-6, IL1B, CCL2, CXCL1, CXCL2, CXCL3, CCL7, C3AR1, SLC16A6, CXCL5 and / or SERPINB2. In some embodiments, compared with mature dendritic cells (e.g., derived from monocytes after culture with LPS), the APC expresses lower levels of CCL22, CSTB, LIPA, CCL17, CCL13, APOE, FABP4, CD1B, FN1, CD1c, CD1A and / or PTGDS. In some embodiments, compared with M1 macrophages, the APC expresses higher levels of CXCL8, IL-6, NAMPT, CXCL1, CCL6, CXCL3, CCL18, PELI1, CXCL5, SLC16A6 and / or SERPINB2. In some embodiments, the APC expresses lower levels of CXCL10, MMP9, LIPA, S100A4, CCL22, IL12B, APOE, CRABP2, PTGDS and / or FN1 compared to M1 macrophages. In some embodiments, the APC expresses higher levels of C3AR1, olr1, TLR2 and PLAUR compared to mature dendritic cells (e.g., monocytes derived from cultured with LPS or TNFα), M1 / M2 macrophages and / or monocytes. In some embodiments, the APC expresses lower levels of DC-specific antigens (e.g., CD11c, CD1a, CD1c, Batf3) compared to mature dendritic cells (e.g., monocytes derived from cultured with LPS or TNFα). In some embodiments, the APC expresses lower levels of macrophage-specific antigens (e.g., CD68) compared to M1 / M2 macrophages. In some embodiments, the APCs express lower levels of monocyte-specific antigens (e.g., CCR2, CXCR1, CD14). In some embodiments, the APCs express lower levels of CD31 compared to monocytes.

[0286] In some embodiments, a population of APCs derived from monocytes obtained from an individual (e.g., a cancer patient or a virally infected patient) is provided, wherein the APCs a) express high levels of one or more antigen presenting molecules, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL and CD40, and / or b) low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from: TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10. In some embodiments, the monocytes obtained from the individual display lower 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) 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.

[0287] In some embodiments, a population of APCs derived from monocytes obtained from an individual (e.g., a cancer patient or a virally infected patient) is provided, wherein the APCs a) express high levels of MHC I, MHC II, CD80, CD40, OX40L (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the expression levels of the corresponding molecules on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF for about 2 days); b) produces high levels (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the expression levels of the corresponding molecules on monocytes obtained from the same individual and cultured with GM-CSF and M-CSF for about 2 days) of IL-12, type I and type II IFN, TNFα, IL-1, and IL-6; c) expresses low levels of TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2), and Siglec 10 (e.g., at least about 10%, 20%, 30%, 40% or 50% lower than the expression level of the corresponding molecule 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.

[0288] In some embodiments, the APCs described herein are derived from monocytes obtained from an individual (e.g., a cancer patient or a virally infected patient). In some embodiments, compared to monocytes obtained from a reference individual (e.g., a healthy individual), the monocytes obtained from the individual display a lower expression level of M-CSFR (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower). In some embodiments, compared to monocytes obtained from a reference individual (e.g., a healthy individual), the monocytes obtained from the individual display a lower expression level of GM-CSFR (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower). In some embodiments, compared to monocytes obtained from a reference individual (e.g., a healthy individual), the monocytes obtained from the individual display a lower expression level of both M-CSFR and GM-CSFR (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower).

[0289] In some embodiments, the monocytes obtained from the individual display a lower expression level of IL-10R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes obtained from the individual display a lower expression level of IL-6R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, the monocytes obtained from the individual display a lower expression level of IL-4R (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) compared to monocytes obtained from a reference individual (e.g., a healthy individual). In some embodiments, monocytes obtained from the individual display lower expression levels of IFNGR (e.g., at least about 10%, 20%, 30%, 40%, 50%, or 60% lower) compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0290] In some embodiments, the APC expresses high levels of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen presenting molecules, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, optionally wherein the APC is generated in cell culture from monocytes (such as any monocytes described herein, e.g., monocytes obtained from a cancer patient). In some embodiments, the APC expresses high levels of MHC I, MHC II, CD86, CD80, CD40, and / or OX40L.

[0291] 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 antigen presenting molecules are selected from the group consisting of: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, wherein the expression level of one or more antigen presenting molecules selected from the group consisting of MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL, and CD40 is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the expression level of the corresponding molecules on monocytes obtained from the same individual and cultured (e.g., for about 2 days) with GM-CSF and M-CSF.

[0292] In some embodiments, the APC expresses high levels of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen presenting molecules, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL, and CD40, wherein the expression level of one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) antigen presenting molecules selected from MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL, and CD40 is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the expression level of the corresponding molecules on dendritic cells obtained from healthy humans and cultured with GM-CSF and IL-4 for about 5 days.

[0293] In some embodiments, the APC expresses high levels of MHC I, MHC II, CD86, CD80, CD40, and / or OX40L.

[0294] 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 type II IFNs, TNFα, IL-1, and IL-6, wherein the expression level of one or more (e.g., at least 2, 3, 4, 5, or 6) cytokines selected from IL-12, type I and type II IFNs, TNFα, IL-1, and IL-6 is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than the expression level of the corresponding cytokines on monocytes obtained from the same individual and cultured (e.g., for about 2 days) with GM-CSF and M-CSF.

[0295] In some embodiments, the APCs produce high levels of PD-L1, wherein the expression level of PD-L1 produced by the APCs obtained from the individual is at least about 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% higher than the expression level of PD-L1 on the monocytes from which they are derived.

[0296] In some embodiments, the APC expresses 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 APC expresses low levels of SIRPα.

[0297] In some embodiments, the APC expresses low levels of inhibitory signaling molecules, where the expression level of one or more antigen presenting molecules selected from TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10 is at least about 10%, 20%, 30%, 40% or 50% lower than the expression level of the corresponding molecules on monocytes obtained from the same individual and cultured (e.g., for about 2 days) using GM-CSF and M-CSF.

[0298] In some embodiments, the APC expresses low levels of inhibitory signaling molecules, and the expression level of one or more antigen presenting molecules selected from TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10 is at least about 10%, 20%, 30%, 40% or 50% lower than the expression level of the corresponding molecules on dendritic cells obtained from healthy individuals and cultured with GM-CSF and IL-4 for about 5 days.

[0299] In some embodiments, the APC has Figure 4 Essentially the same morphology is shown in F (right).

[0300] In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the APCs have dendritic cell morphology.

[0301] In some embodiments, the APC comprises one or more tumor-associated antigenic peptides, e.g., neoantigenic peptides (e.g., any of those discussed herein).

[0302] In some embodiments, the APC comprises one or more virus-associated antigenic peptides.

[0303] In some embodiments, the APC can promote the proliferation of immune cells (e.g., T cells, e.g., CD4 T cells and / or CD8 T cells) after being cultured with immune cells. In some embodiments, the APC promotes the proliferation of T cells at least about 5 times, 10 times, 15 times, or 20 times in a culture comprising IL-2, IL-7, and IL-15. In some embodiments, the culture is no longer than about 24 hours, 22 hours, 20 hours, or 18 hours. In some embodiments, the APC presents one or more disease-associated peptides (e.g., tumor peptides) to immune cells.

[0304] In some embodiments, a composition (eg, culture) is provided, comprising an APC as described herein. In some embodiments, the APC presents one or more disease-associated peptides (eg, tumor peptides) to immune cells.

[0305] Tumor-associated antigen peptide

[0306] Various methods can be used to identify tumor-associated antigenic peptides.

[0307] One method commonly used to determine the peptides recognized by the CTL is to perform expression cloning, which consists in isolating the peptide encoding gene by transfecting a tumor cDNA library and testing the transfected cells for their ability to activate CTL clones. Fragments of the determined gene can then be transfected to define the region encoding the antigenic peptide, and finally the candidate peptide with the appropriate HLA binding motif can be tested for its ability to sensitize target cells to lysis by CTL. This method has been successfully used to determine a large number of antigenic peptides.

[0308] Today, tumor-associated antigenic peptides are often identified using a "reverse immunology" approach that involves selecting peptides with appropriate HLA binding motifs within a target protein (e.g., proteins encoded by mutated oncogenes or by genes selectively expressed or overexpressed by tumors). Candidate peptides are synthesized and tested for HLA binding in vitro. The most effective binders are pulsed onto antigen-presenting cells, which are used to stimulate T lymphocytes in vitro to derive CTL lines or clones that recognize target cells that are hit by the peptide. The disadvantage of this approach is that the recognized peptides may not be effectively processed by the tumor. Therefore, it is necessary to verify that CTLs do recognize tumor cells that naturally express the peptide-encoding gene. In addition, transfectants with normal gene expression levels, or cells in which the expression of the gene has been knocked down using si or shRNA, should be tested.

[0309] A third approach to identifying antigens is to elute antigenic peptides based on MHC class I molecules immunopurified from the surface of tumor cells. Direct identification of the sequence of eluted peptides by mass spectrometry is technically demanding but has proven useful for identifying or confirming the relevance of peptides that have undergone post-translational modifications such as serine / threonine phosphorylation, glycosylation-dependent asparagine deamidation, or peptide splicing.

[0310] These various methods have been used to identify a large number of antigenic peptides recognized by anti-tumor CTLs. These antigens can be conveniently classified according to the expression pattern of the parental gene. A regularly updated database of those antigenic peptides effectively presented by tumor cells can be found at the http: / / www.cancerimmunity.org / website. See Vigneron, Biomed Res Int. 2015; 2015:948501.

[0311] The present application provides APCs prepared by any of the methods described herein.

[0312] New antigen peptide

[0313] There are various methods for detecting and screening new antigens. Sandwich immunoassays of miniaturized systems can 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 protein blotting (western blot) consists of an isoelectric focusing (IEF) gel running in the first dimension and an SDS-PAGE gel running in the second dimension. SERPA separates the proteins in the gel by their isoelectric point (IP) and molecular weight, and then transfers the proteins from the gel to a carrier membrane to screen antibodies. Finally, the antigen protein spots can be identified by MS. See, for example, Tjalsma et al., Proteomics Clin.Appl.2 167-180 (2008). This method has been used to identify antigens in different tumor types. Serological analysis of recombinant cDNA expression libraries (SEREX), which combines serological analysis with antigen cloning technology, is a widely used technology for exploring the antigen library of tumors. SEREX first constructs a cDNA library from a cancer cell line or a fresh tumor sample, then screens the cDNA library with the autologous serum of a cancer patient, and finally sequences the immunoreactive clones. SEREX has identified various tumor antigens, including CTA, 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 multiple affinity protein profiling / MAPPing and nanoplasma biosensors) have also been developed to identify tumor antigens. See, for example, Lee et al., Biosens. Bioelectron. 74 341-346 (2015).

[0314] In some embodiments, one or more neoantigenic peptides described herein are obtained from a neoantigen database (such as any of the neoantigen databases described herein). For example, Tan et al. constructed a manually curated database of human tumor neoantigenic peptides ("dbPepNeo") based on the following four criteria: (i) the peptide is isolated from human tumor tissue or cell line, (ii) the peptide contains non-synonymous mutations in the amino acid sequence, (iii) the peptide can be bound by HLA-I molecules, and (iv) the peptide can induce CD8+T cell response. See Tan et al., Database (Oxford). January 1, 2020; 2020: baaa004. Xia et al. constructed another database NEPdb, which uses a state-of-the-art predictor to provide pan-cancer-level predicted HLA-I neoepitopes derived from 16,745 common cancer somatic mutations. See Xia et al., Front Immunol. 2021; 12: 644637. Wu et al. developed a comprehensive tumor-specific new antigen database (TSNAdb v1.0) based on pan-cancer immunogenomic analysis of somatic mutation data and human leukocyte antigen (HLA) allele information from 7748 tumor samples in 16 tumor types from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). See Wu et al., enomics proteomics bioinformatics. 2018 August; 16 (4): 276-282.

[0315] In some embodiments, the one or more neoantigenic peptides are obtained from analyzing the biological information of an individual (such as a patient with cancer). In some embodiments, the neoantigenic peptides are obtained from a computer analysis of the tumor genome of a cancer patient. See, for example, Roudko et al., Front Immunol. 2020; 11: 27. In some embodiments, the neoantigenic peptides are obtained from a computer analysis of the transcriptome of a cancer patient. See, for example, Caushi et al., Nature. 2021 August; 596(7870): 126-132. In some embodiments, the neoantigenic peptides are obtained from a computer analysis of the proteome of a cancer patient. See, for example, Wen et al., Nat Commun. 2020 April 9; 11(1): 1759.

[0316] In some embodiments, the neoantigenic peptide is selected based on patient data. In some embodiments, the patient data is derived from data from a group of patients suffering from 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 suffering from any cancer. In some embodiments, the group of patients is from the same sex (e.g., male or female). In some embodiments, the group of patients is from the same race. In some embodiments, the group of patients has one or more biomarkers (e.g., a specific gene (e.g., KRAS, e.g., PTEN) abnormal).

[0317] In some embodiments, the one or more new antigenic peptides are derived from any polypeptide known or found to contain tumor-specific mutations. Suitable polypeptides from which new antigenic peptides can be derived can be found in, for example, various databases available in this field (e.g., COSMIC database). These databases manage comprehensive information on somatic mutations in human cancers. In some embodiments, the peptide contains tumor-specific mutations. In some embodiments, the tumor-specific mutation is a driver mutation for a specific cancer type.

[0318] In some embodiments, the tumor-associated peptide (eg, a new antigenic peptide) is a synthetic peptide. In some embodiments, the new antigenic peptide is obtained by exome high-throughput sequencing and pre-screened using an epitope prediction algorithm.

[0319] In some embodiments, the one or more neoantigenic peptides are selected based on their binding affinity to MHC molecules (e.g., MHC I molecules and / or MHC II molecules). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of less than 5000nM (e.g., less than 500nM, less than 250nM, less than 100nM, or less than 50nM) (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of about 500-5000nM (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of less than 500nM (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of about 250-500nM IC50. In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of less than 250nM (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of less than 100 nM (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of about 50-500 nM IC50. In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of less than 50 nM (IC50). In some embodiments, the neoantigenic peptide has a binding affinity to the MHC molecule of about 1-50 nM IC50.

[0320] In some embodiments, a plurality of tumor-associated peptides (eg, neoantigenic peptides) are prepared from surgical resections of tumor tissue or biopsy extracts thereof.

[0321] In some embodiments, a plurality of tumor-associated peptides (eg, neoantigenic peptides) are prepared from a mixture of tumor cells or extracts thereof isolated from tumor tissue or biopsies.

[0322] In some embodiments, a plurality of tumor-associated peptides (eg, neo-antigenic peptides) are prepared from a mixture of isolated tumor-associated peptides (eg, neo-antigenic peptides).

[0323] In some embodiments, the above tumor tissue or cell is fresh tumor tissue or cell.In some embodiments, the tumor tissue or cell is obtained from a frozen sample.

[0324] In some embodiments, the tumor tissue or cells have undergone induction of immunogenic cell death (eg, freeze-thaw to lyse tumor cells, high dose UV irradiation, X-ray irradiation).

[0325] In some embodiments, the tumor tissue or cells have been subjected to radiation therapy.

[0326] IV. Methods for activating immune cells and immune activating compositions

[0327] The application also provides a method for activating an immune cell population. In some embodiments, the method includes co-culturing the immune cell population with an APC population described herein, wherein the APC is preloaded with one or more antigenic peptides (e.g., tumor peptides, such as tumor-associated peptides, e.g., new antigenic peptides).

[0328] In some embodiments, a method for activating a group 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 immune cell group with an APC group (e.g., an APC described herein), thereby producing an activated immune cell group, wherein the APC is pre-loaded with one or more tumor peptides. In some embodiments, the APC is derived from monocytes obtained from the same individual. In some embodiments, the APC has been pre-cultured with a tumor antigen (e.g., frozen-thawed tumor cells / fragments). In some embodiments, the pre-culture is about 3-10 hours (e.g., about 6 hours). In some embodiments, during co-culture, the ratio of APC to immune cells (e.g., T cells, e.g., TIL cells) 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 APC and immune cells are co-cultured for about 2-20 days. In some embodiments, IL-2, IL-7 and / or IL-15 are supplemented to the co-culture (e.g., supplemented for about at least 2 days or 3 days after co-culture). In some embodiments, the activated immune cells comprise 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 activated by co-culturing with the APCs described herein for at least two, three, four or five rounds. In some embodiments, after two, three or four consecutive rounds (e.g., about 6-10 days per round) of activation involving co-culture described herein, the activated immune cells do not show exhaustion characteristics (e.g., aging). In some embodiments, the co-culture does not involve the use of anti-CD3 antibodies and / or anti-CD28 antibodies in at least some rounds (e.g., anti-CD3 and anti-CD28 antibodies are used only in the first round, not in subsequent rounds).

[0329] In some embodiments, the method includes contacting the APC with a composition comprising a plurality of tumor-associated peptides (e.g., neoantigenic peptides). In some embodiments, the APC is allowed to contact the composition comprising a plurality of tumor-associated peptides (e.g., neoantigenic peptides) for about 4 hours to about 24 hours.

[0330] In some embodiments, the APC has been pre-cultured with a composition comprising a plurality of tumor-associated peptides (eg, neoantigenic peptides).

[0331] In some embodiments, the composition comprising a plurality of tumor-associated peptides (eg, neoantigenic peptides) is a surgical resection of a tumor tissue or a biopsy extract thereof.

[0332] In some embodiments, the composition comprising a plurality of tumor-associated peptides (eg, neoantigenic peptides) is a mixture of tumor cells isolated from tumor tissue or biopsy or extracts thereof.

[0333] In some embodiments, the composition comprising a plurality of tumor-associated peptides (eg, neoantigenic peptides) is a mixture of isolated tumor-associated peptides (eg, neoantigenic peptides).

[0334] In some embodiments, the tumor tissue or cell is a fresh tumor tissue or cell. In some embodiments, the tumor tissue or cell is obtained from a frozen sample.

[0335] In some embodiments, the tumor tissue or cell has undergone apoptosis induction.

[0336] In some embodiments, the tumor tissue or cells have been subjected to radiation therapy.

[0337] In some embodiments, the population of immune cells and APCs are derived from the same individual.

[0338] In some embodiments, the population of immune cells and the antigen presenting cells are not derived from the same individual.

[0339] The present application also provides activated immune cells (eg, T cells) prepared by any of the methods described herein.

[0340] Tumor-associated peptide loading

[0341] In some embodiments, the methods described herein further include contacting the APC with a plurality of tumor-associated peptides (e.g., neoantigenic peptides). In some embodiments, the plurality of tumor-associated peptides (e.g., neoantigenic peptides) have more than about 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., neoantigenic peptides) in any one. In some embodiments, the APC is allowed to contact the composition comprising a plurality of tumor-associated peptides (e.g., neoantigenic peptides) for about 4 hours to about 24 hours.

[0342] In some embodiments, the APCs have been pre-cultured with a composition comprising a plurality of tumor-associated peptides (eg, neoantigenic peptides) prior to use in the methods for activating immune cells described herein.

[0343] An exemplary embodiment of contacting an APC population with a plurality of tumor-associated peptides (e.g., neoantigenic peptides) comprises pulsing a plurality of tumor-associated peptides (e.g., neoantigenic peptides) into the APC population. As known in the art, "pulsing" refers to a process in which a cell (e.g., APC) is mixed with a solution containing a tumor-associated peptide (e.g., neoantigenic peptide), and optionally subsequently removing the tumor-associated peptide (e.g., neoantigenic peptide) from the mixture. The APC population may be contacted with a plurality of tumor-associated peptides (e.g., neoantigenic peptides) for a few seconds, a few minutes, or a few 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, a week, 10 days, or any one of a longer period of time. The concentration of each neoantigenic peptide used in the contacting step may 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., neoantigenic peptide) is about 0.1-200 μg / mL, including, for example, about 0.1-0.5, 0.5 to 1, 1-10, 10-50, 50-100, 100-150, or 150-200 μg / mL.

[0344] In some embodiments, the APC population is contacted with a plurality of tumor-associated peptides (e.g., neoantigenic peptides) in the presence of a composition that promotes the uptake of a plurality of tumor-associated peptides (e.g., neoantigenic peptides) by APCs. In some embodiments, the compound, material, or composition may be contained in a solution that promotes a plurality of tumor-associated peptides (e.g., neoantigenic peptides) to promote peptide uptake by APCs. Compounds, materials, or compositions that promote the uptake of a plurality of tumor-associated peptides (e.g., neoantigenic peptides) by APCs include, but are not limited to: lipid molecules and peptides having a plurality of positively charged amino acids. In some embodiments, the APC population takes up more than about 50%, 60%, 70%, 80%, 90%, or 95% of any one of the tumor-associated peptides (e.g., neoantigenic peptides). In some embodiments, more than about 50%, 60%, 70%, 80%, 90%, or 95% of the APCs in the APC population take up at least one tumor antigen peptide.

[0345] Immune cells

[0346] The immune cells described herein may be any type of immune cells that interact with APCs and can be activated by APCs and then exert their desired functions. Exemplary immune cells include T cells.

[0347] T cells or T lymphocytes play a central role in cell-mediated immunity. Each clone of activated T cells expresses a different T-cell receptor (TCR) on the surface, which is responsible for recognizing 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 different functions.

[0348] Cytotoxic T cells (TC) participate in the immune response to tumor cells and other infected cells (such as virus-infected cells), and destroy tumor cells and other infected cells. In general, TC cells work by recognizing class I MHC-presented antigens on APC or any target cell. TCR together with co-stimulators (e.g., CD28 on T cells bound to B7 on APC, or by helper T cell stimulation) stimulation leads to the activation of TC cells. The activated TC cells can then proliferate and release cytotoxins, thereby destroying APC or target cells (such as cancer cells). Mature TC cells generally express surface proteins CD3 and CD8. Cytotoxic T cells belong to CD3+CD8+T cells.

[0349] Helper T cells (TH) are T cells that help the activity of other immune cells by releasing T cell cytokines, which can regulate or inhibit immune responses, induce cytotoxic T cells, and maximize the cell killing activity of macrophages. In general, TH cells work by recognizing class II MHC-presented antigens on APCs. Mature TH cells express surface proteins CD3 and CD4. Helper T cells belong to CD3+CD4+T cells.

[0350] Regulatory T cells (T REG T cells generally regulate the immune system by promoting tolerance to self-antigens and thus limiting autoimmune activity. REG Promotes cancer cells to escape from immune response. REG T cells generally express CD3, CD4, CD7, CD25, CTLA4, GITR, GARP, FOXP3 and / or LAP. CD4+CD25+Foxp3+T cells are a type of T REG cell.

[0351] Memory T cells (Tm) are T cells that have previously encountered and responded to their specific antigens, or T cells that have differentiated from activated T cells. Although tumor-specific Tm constitute a small part of the total amount of T cells, it plays a key role in monitoring tumor cells during a person's lifetime. If tumor-specific Tm encounters tumor cells expressing its specific tumor antigen, the Tm is immediately activated and cloned and expanded. Activated and amplified T cells differentiate into effector T cells, killing tumor cells with high efficiency. Memory T cells are important for establishing and maintaining long-term tumor antigen-specific responses of T cells.

[0352] Typically, the antigen of a T cell is a protein molecule, or a linear fragment of a protein molecule that can be recognized by a T-cell receptor (TCR) to cause a specific T cell response. The antigen can be derived from an exogenous source (such as a virus-encoded protein), or an endogenous source (such as a protein expressed on the cell surface). The smallest fragment of an antigen that is directly involved in interacting with a specific TCR is called an epitope. Multiple epitopes can be present in a single antigen, where each epitope is recognized by a different TCR encoded by a specific clone of a T cell.

[0353] In order to be recognized by TCR, antigenic peptides or antigenic fragments are processed into epitopes by APC (such as dendritic cells), and then combined in an elongated configuration inside the major histocompatibility (MHC) molecule to form an MHC-peptide complex on the surface of APC (such as dendritic cells). MHC molecules are also called human leukocyte antigens (HLA). MHC provides an expanded binding surface for the strong association between TCR and epitopes, while the combination of unique amino acid residues in the epitope ensures the specificity of the interaction between TCR and the epitope. Based on its structural features, human MHC molecules are divided into two types: MHC class I and MHC class II, especially based on the length of the epitope bound inside the corresponding MHC complex. MHC-I epitopes are epitopes that are bound to and represented by MHC class I molecules. MHC-II epitopes are epitopes that are bound to and represented by MHC class II molecules. MHC-I epitopes are usually about 8 to about 11 amino acids long, while MHC-II epitopes are about 13 to about 17 amino acids long. Due to genetic polymorphism, various subtypes of both MHC class I molecules and MHC class II molecules exist in the human population. The response of T cells to specific antigenic peptides presented by MHC class I or MHC class II molecules on APCs is called MHC-restricted T cell response.

[0354] In some embodiments, the immune cells are selected from the group consisting of: PBMCs, tumor infiltrating T cells (TILs), and T cells (e.g., CD4 T cells and / or CD8 T cells).

[0355] In some embodiments, the immune cell is PBMC.

[0356] In some embodiments, the immune cell is a tumor infiltrating T cell (TIL).

[0357] In some embodiments, the immune cell is a CD4 T cell and / or a CD8 T cell.

[0358] In some embodiments, the immune cell and the APC are derived from the same individual. In some embodiments, the immune cell and the APC are derived from different individuals.

[0359] Preparation of activated immune cells (eg, activated T cells)

[0360] The methods described herein include co-culturing a population of immune cells (eg, T cells) with a population of APCs loaded with a plurality of tumor-associated peptides (eg, neoantigenic peptides) as described herein.

[0361] In some embodiments, the co-culture is performed for at least 24 hours. In some embodiments, the co-culture is performed for at least about 1-5 days (e.g., about 1-3 days). In some embodiments, the immune cell (e.g., T cell) group is co-cultured with an APC group loaded with a variety of tumor-associated peptides (e.g., neoantigenic peptides) for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 days. In some embodiments, the immune cell (e.g., T cell) group is co-cultured with an APC group loaded with a variety of tumor-associated peptides (e.g., neoantigenic peptides) for about 14 to about 21 days. In some embodiments, the immune cell (e.g., T cell) group is co-cultured with an APC group loaded with a variety of tumor-associated peptides (e.g., neoantigenic peptides) for about 14 days.

[0362] Immune cell (for example, T cell) group in any embodiment of the method described herein can be derived from various sources. A convenient source of immune cells is PBMC from human peripheral blood. For example, T cell group can be separated from PBMC, or PBMC group enriched with T cells (such as by adding T cell-specific antibodies and cytokines) can be used in co-culture. In some embodiments, T cell group used in co-culture is obtained from the non-adherent part of peripheral blood mononuclear cells (PBMC). In some embodiments, the PBMC is obtained by density gradient centrifugation of peripheral blood samples. In some embodiments, the activated T cell group is obtained by obtaining a non-adherent PBMC group, and the non-adherent PBMC group is co-cultured with an APC group loaded with a variety of tumor-associated peptides (for example, neoantigen peptides) (for example, in the presence of at least one cytokine (such as IL-2) and anti-CD3 antibodies) to prepare.

[0363] The co-culture may also contain cytokines and other compounds to promote activation, maturation and / or proliferation of T cells, as well as to induce the later differentiation of T cells into (for example) memory T cells. Exemplary cytokines that can be used in this step include (but are not limited to): IL-7, IL-15, IL-21, etc. Certain cytokines can help inhibit T cells in the activated T cell population in the co-culture. REG For example, in some embodiments, a high dose (e.g., about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, or 1500 U / ml) of a cytokine (e.g., IL-2) is used to co-culture a population of T cells with a population of dendritic cells loaded with a variety of tumor antigen peptides to obtain a population of T cells with a low percentage of T cells. REG An activated T cell population of cells.

[0364] In some embodiments, the method of activating immune cells comprises co-culturing immune cells (e.g., T cells) and APC populations for more than one round (e.g., 2, 3, or 4 rounds). In some embodiments, each round takes about 6 to 8 days. In some embodiments, the first, second, third, and / or fourth rounds do not involve the addition of anti-CD3 antibodies and / or anti-CD28 antibodies. In some embodiments, the immune cells (e.g., T cells) show non-exhausted characteristics after 2, 3, or 4 rounds of co-culture. In some embodiments, the immune cells (e.g., T cells) can expand by about 50-100 times (e.g., at least 50 times) 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) reaches about 10 after three or four rounds of co-culture. 10 indivual.

[0365] In some embodiments, the method of activating immune cells described herein further comprises expanding the immune cell population after the co-culturing step. In some embodiments, expanding the immune cell population comprises contacting the immune cells with a cytokine selected from IL-2, IL-7, and IL-15, optionally for about 2 to about 10 days. In some embodiments, the co-culturing is in the presence of an anti-CD3 antibody and a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof).

[0366] The present application also provides activated immune cell populations obtained by the methods described in this section.

[0367] V. Treatment Methods

[0368] The present application also provides a method of treating a disease or condition (such as cancer, for example, viral infection) in a patient, comprising administering to the patient a population of APCs and / or activated immune cells obtained by the above method.

[0369] In some embodiments, a method of treating a disease or condition (such as cancer, e.g., a viral infection) in a patient is provided, comprising administering to the patient a population of APCs (such as any of those described in Section III or produced according to the methods described in Section II).

[0370] In some embodiments, a method of treating a disease or condition (such as cancer, e.g., 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 virally infected patient), wherein the APCs a) express high levels of one or more antigen presenting molecules, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL and CD40, and / or b) low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from: TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10. In some embodiments, compared to monocytes obtained from a reference individual (e.g., a healthy individual), monocytes obtained from the individual display 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). In some embodiments, the method further comprises administering a second therapy that induces immunogenic cell death (e.g., radiotherapy). In some embodiments, the method comprises administering APC and radiotherapy in combination, simultaneously or sequentially. In some embodiments, prior to administration, the APC has not been preloaded with a disease or condition-associated antigen (e.g., a tumor antigen or a viral antigen). In some embodiments, prior to administration, the APC has been preloaded with a disease or condition-associated antigen (e.g., a tumor antigen or a viral antigen).

[0371] In some embodiments, a method of treating a disease or condition (such as cancer, for example, 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 virally infected patient), wherein the APCs are obtained by the following steps: a) contacting a population of monocytes obtained from an individual with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining a population of APCs, wherein the plurality of S / D / M factors include: 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 method further comprises administering a second therapy that induces immunogenic cell death (e.g., radiation therapy). In some embodiments, the method comprises administering APCs and radiation therapy in combination, simultaneously, or sequentially. In some embodiments, prior to administration, the APC has not been preloaded with a disease or condition-associated antigen (e.g., a tumor antigen or a viral antigen). In some embodiments, prior to administration, the APC has been preloaded with a disease or condition-associated antigen (e.g., a tumor antigen or a viral antigen).

[0372] In some embodiments, a method of treating a disease or condition (such as cancer, e.g., 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 virally infected patient), wherein the APCs a) express high levels of one or more antigen presenting molecules, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL and CD40, and / or b) low levels of inhibitory signaling molecules, wherein the inhibitory signaling molecules are selected from: TGFβR, SIRPα, LILRB (LILRB1 and / or LILRB2) and Siglec 10, wherein prior to administration, the APCs have been pre-loaded with a disease or condition associated antigen (e.g., a tumor antigen or a viral antigen). In some embodiments, monocytes obtained from the individual 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) compared to monocytes obtained from a reference individual (e.g., a healthy individual).

[0373] In some embodiments, a method of treating a disease or condition (such as 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 have undergone co-culture with a population of APCs, wherein the APCs are generated after contact with an IL-10 receptor activator (IL-10R activator) and one or more of an IFNγ receptor activator (IFNR activator), a TNFα receptor activator (TNFR activator), and an IL-4 receptor activator (IL-4R activator), and wherein prior to co-culture, the APCs have been pre-loaded with one or more peptides associated with the disease or condition (e.g., tumor-associated peptides, e.g., neoantigenic peptides, virus-specific peptides).

[0374] In some embodiments, a method of treating a disease or condition (such as 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), wherein the immune cells have undergone co-culture with a population of APCs, wherein the APCs are produced after contact with IL-10 and one or more of IFNγ, TNFα, and IL-4, and wherein prior to co-culture, the APCs have been pre-loaded with one or more peptides associated with the disease or condition (e.g., tumor-associated peptides, e.g., neoantigenic peptides, virus-specific peptides). In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are T cells obtained from the patient's peripheral blood (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). In some embodiments, the immune cell is a T cell (e.g., a CD3 T cell, e.g., a CD4 T cell, e.g., a CD8 T cell, e.g., both CD4 and CD8 T cells, e.g., TIL) obtained from the peripheral blood of an individual different from the patient (optionally with a matching HLA type). In some embodiments, the APC and the activated immune cell are derived from the same individual. In some embodiments, the APC and the activated immune cell are derived from different individuals (optionally with a matching HLA type). In some embodiments, the APC is produced after contact with IL-10, IFNγ, TNFα, and IL-4. In some embodiments, the APC is produced after contact with IL-10, IFNγ, TNFα, GM-CSF, IL-6, and IL-4. In some embodiments, the APC is produced after contact with one or more of the optimization factors described in Section II. In some embodiments, the activated immune cell is administered intratumorally, intraperitoneally, or intravenously. In some embodiments, the activated immune cell is administered at about 10 7 Up to 10 9In some embodiments, the methods of treatment described herein further include treating the patient with chemotherapy, radiotherapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with irradiation. In some embodiments, the irradiation site is different from the site of the cancer to be treated.

[0375] In some embodiments, a method for treating a virus-associated cancer in a patient is provided, comprising administering to the patient an activated T cell population, wherein the T cells have undergone co-culture with an APC population, wherein the APC is produced after contact with one or more of IL-10 and IFNγ, TNFα, and IL-4, and wherein prior to co-culture, the APC has been pre-loaded with one or more tumor-associated peptides (e.g., neoantigenic peptides) associated with virus-associated cancer, wherein viral antigen-reactive T cells have been removed from the activated T cell population prior to administration. In some embodiments, the APC is derived from a patient. In some embodiments, the activated T cells are derived from a patient. In some embodiments, both the APC and the activated T cells are derived from a patient. 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 7 Up to 10 9 In some embodiments, the methods of treatment described herein further include treating the patient with chemotherapy, radiotherapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with radiation. In some embodiments, the radiation site is different from the site of the cancer to be treated.

[0376] In some embodiments, a method for treating a patient's liver cancer associated with a virus (e.g., hepatitis B virus or hepatitis C virus) is provided, comprising administering to the patient an activated T cell population, wherein the T cells have undergone co-culture with an APC population, wherein the APC is produced after contact with one or more of IL-10 and IFNγ, TNFα, and IL-4, and wherein prior to co-culture, the APC has been pre-loaded with one or more tumor-associated peptides (e.g., neoantigenic peptides), wherein viral antigen-reactive T cells have been removed from the activated T cell population prior to administration. In some embodiments, the APC is derived from a patient. In some embodiments, the activated T cells are derived from a patient. In some embodiments, both the APC and the activated T cells are derived from a patient. 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 7 Up to 10 9In some embodiments, the methods of treatment described herein further include treating the patient with chemotherapy, radiotherapy, or an immune checkpoint inhibitor. In some embodiments, the method includes treating the patient with radiation. In some embodiments, the radiation site is different from the site of the cancer to be treated.

[0377] patient

[0378] In some embodiments, the patient has a solid tumor. In some embodiments, the patient has a blood cancer.

[0379] In some embodiments, the patient has advanced cancer. In some embodiments, the patient has late stage cancer. In some embodiments, the patient has cancer in stage II, III, or IV. In some embodiments, the patient has an inoperable tumor and / or metastasis. In some embodiments, the patient is a terminal disease patient.

[0380] In some embodiments, the patient is female. In some embodiments, the patient is male.

[0381] 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 old.

[0382] cancer

[0383] As discussed above, therapeutic methods involving immune cells (eg, T cells) activated by APCs generated by the various methods described herein are applicable to all types of cancer.

[0384] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood cancer.

[0385] 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 malignant.

[0386] In some embodiments, the cancer has been treated with one or more prior therapies (e.g., immune checkpoint blockade therapy (e.g., PD-1 antibody), chemotherapy, surgery, cellular therapy (e.g., allogeneic NK cell infusion therapy)), and / or has failed to respond to the prior therapies.

[0387] In some embodiments, the cancer is a relapsed or refractory cancer.

[0388] Examples of cancers described herein include, but are not limited to, adrenocortical carcinoma, myeloid metaplasia of unknown etiology, 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., fibrillary astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive ectodermal tumors, optic pathway and hypothalamic gliomas and glioblastomas), breast cancer, bronchial adenoma / carcinoid, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), cancer of unknown primary, 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 tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., liver cancer and hepatoma), hypopharyngeal cancer, pancreatic islet cell carcinoma, Cancer of the lining of the lung (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), lymphoid tumors (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., epithelial ovarian cancer, ovarian germ cell tumor, ovarian tumor of low malignant potential), pancreatic cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastoma, lymphoma, primary central neurocytic lymphoma (microglioma), pulmonary lymphangioleiomyomatosis, colorectal cancer, kidney cancer, renal pelvis and ureteral cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms tumor and post-transplantation lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with macular disease, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0389] In some embodiments, the cancer is a viral infection-related cancer. In some embodiments, the cancer is a human papillomavirus (HPV)-related cancer (e.g., HPV-related cervical cancer, e.g., HPV-related head and neck cancer, e.g., HPV-related squamous cell carcinoma). In some embodiments, the cancer is a human herpesvirus 8 (HHV8)-related cancer (e.g., Kaposi's sarcoma). In some embodiments, the cancer is a human T-lymphotrophic virus (HTLV-1)-related cancer (e.g., adult T-cell leukemia or lymphoma). In some embodiments, the cancer is an Epstein-Barr virus (EBV)-related cancer (e.g., Burkitt's lymphoma, Hodgkin's and non-Hodgkin's lymphoma, gastric cancer). In some embodiments, the cancer is a hepatitis B virus (HBV)-related cancer (e.g., liver cancer). In some embodiments, the cancer is a hepatitis C virus-related cancer (e.g., liver cancer, non-Hodgkin's lymphoma).

[0390] Dosages and methods of administering activated immune cells (eg, T cells)

[0391] The activated immune cells can be administered at any desired dose, which in some aspects includes the desired dose or number of cells or cell type. Thus, in some embodiments, the dose of the cell is based on the total number of cells (or number / kg body weight) and / or the desired ratio of a single population. In some embodiments, the dose of the cell is based on the desired total number (or number / kg body weight) of cells or a single cell type in a single population.

[0392] In certain embodiments, the amount of activated immune cells (e.g., T cells, e.g., CD4 and / or CD8 T cells, e.g., TILs) administered to an individual is in the range of about 1 million to about 100 billion cells and / or the amount of the cells / kg body weight, such as 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 above values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 6 million cells, or a range defined by any two of the above values). In some cases, the dosage may be about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 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 above values), and in some cases, an amount of about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 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 above values), and in some cases, an amount of about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 90 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges and / or per kilogram of body weight. Dosages may vary depending on the specific attributes of the disease or disorder and / or patient and / or other treatments.

[0393] In some embodiments, for example, in the case of a human subject, a dose comprises less than about 1 x 10 9 Total activated immune cells, for example, about 1x10 6 Up to 5x10 8 The range of these cells is, for example, 2x10 6 , 5x10 6 , 1x10 7 , 5x10 7 , 1x10 8 , 5x10 8 or 1x10 9 These cells, or a range between any two of the above values.

[0394] In some embodiments, the activated immune cells are expressed at about 10 7 Up to 10 9 cells / dose administered.

[0395] In some embodiments, the method of treatment comprises administering a total of about 1x10 6 Up to 1x10 9 (For example, 10 6 Up to 10 7 , 107 Up to 10 8 , or 10 8 Up to 10 9 ) activated immune cells (e.g., total CD3 T cells, both CD4 and CD8 T cells, only CD4 T cells, only CD8 T cells, or TILs).

[0396] In some embodiments, the dose of activated immune cells is administered to the subject as a single dose, or is administered only once over a period of two weeks, one month, three months, six months, one year, or longer.

[0397] In some embodiments, the total activated immune cell dose is between 10 4 or about 10 4 with 10 9 or about 10 9 cells / kg body weight, such as 10 5 with 10 6 cells / kg body weight, for example, about 1x10 5 cells / kg body weight, 1.5x10 5 cells / kg body weight, 2x10 5 cells / kg body weight, or 1x10 6 Cells / kg body weight. For example, in some embodiments, the activated immune cells are administered in an amount of: 10 4 or about 10 4 with 10 9 or about 10 9 T cells / kg body weight (e.g. 10 5 with 10 6 T cells / kg body weight) or within a certain error range thereof, for example, 1x10 or about 1x10 5 T cells / kg body weight, 1.5x10 5 T cells / kg body weight, 2x10 5 T cells / kg body weight, or 1x10 6 T cells / kg body weight.

[0398] In some embodiments, the activated immune cells are administered in an amount of: 10 4 or about 10 4 with 10 9 or about 10 9 CD4+ and / or CD8+ cells / kg body weight (e.g. 10 5 with 10 6 CD4+ and / or CD8+ cells / kg body weight) or within a certain error range thereof, for example, 1x10 or about 1x105 CD4+ and / or CD8+ cells / kg body weight, 1.5x10 5 CD4+ and / or CD8+ cells / kg body weight, 2x10 5 CD4+ and / or CD8+ cells / kg body weight, or 1x10 6 CD4+ and / or CD8+ cells / kg body weight.

[0399] In some embodiments, the activated immune cells are present in an amount greater than and / or at least about 1 x 10 6 , about 2.5x10 6 , about 5x10 6 , about 7.5x10 6 or about 9x10 6 CD4+ cells, and / or at least about 1x10 6 , about 2.5x10 6 , about 5x10 6 , about 7.5x10 6 or about 9x0 6 CD8+ cells, and / or at least about 1x10 6 , about 2.5x10 6 , about 5x10 6 , about 7.5x10 6 or about 9x10 6 In some embodiments, the activated immune cells are administered in an amount between about 10 8 with 10 12 Between or about 10 10 with 10 11 T cells, between about 10 8 with 10 12 Between or about 10 10 with 10 11 CD4+ cells, and / or between about 10 8 with 10 12 Between or about 10 10 with 10 11 The amount of CD8+ cells should be between 1 and 20 μg / mL or within a certain error range thereof.

[0400] For the prevention or treatment of a disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, whether the activated immune cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to activated immune cells, and the discretion of the attending physician. In some embodiments, the compositions and cells are suitable for administration to a subject once or over a series of treatments.

[0401] In some embodiments, the APCs described herein are present at about 5000 to about 10,000 cells / mm 3 / range of tumor mass is administered to a subject.

[0402] In some aspects, the size of the dose is determined based on one or more criteria, such as the subject's response to prior treatment (e.g., chemotherapy), the subject's disease burden (e.g., tumor burden, volume, size, or extent), the extent or type of metastasis, the stage and / or likelihood or incidence of the subject developing toxic consequences (e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or a host immune response to the activated immune cells being administered).

[0403] In some aspects, the dose size is determined by the burden of the disease or condition of the subject. For example, in some aspects, the number of cells administered with a dose is determined based on the tumor load present in the subject before the initial dose of cells is administered. In some embodiments, the size of the first and / or subsequent doses is negatively correlated with the burden of disease. In some aspects, such as in the context of a large burden of disease, a small number of cells are administered to the subject. In other embodiments, such as in the context of a lower burden of disease, a greater number of cells are administered to the subject.

[0404] The activated immune cells can be administered by any suitable method, for example, by bolus, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, subtenon fascia (Tenon's) injection, retrobulbar injection, peribulbar injection or near scleral delivery. In some embodiments, it is administered parenterally, intrapulmonary and intranasally, and if required for local treatment, then intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus of activated immune cells. In some embodiments, it is administered by multiple bolus of activated immune cells, for example, over a period of no more than 3 days, or by continuous infusion of activated immune cells.

[0405] In some embodiments, the activated immune cells are administered intratumorally, intraperitoneally, or intravenously.

[0406] Combination therapy

[0407] In some embodiments, the APC (e.g., antigen-stimulated or initial) or activated immune cells are administered simultaneously, combined or sequentially as part of a combination therapy, such as with another therapeutic intervention (i.e., a second therapy), such as an antibody or an engineered cell or receptor or agent (such as a cytotoxic agent or therapeutic agent). In some embodiments, the APC or activated immune cells are administered before another therapeutic intervention. In some embodiments, the APC or activated immune cells are administered after another therapeutic intervention. In some embodiments, the APC or activated immune cells are administered together with one or more additional therapeutic agents, or in combination with another therapeutic intervention, simultaneously, combined or sequentially in any order. In some contexts, the APC or activated immune cells are administered together with another therapy in a sufficiently close time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the APC or activated immune cells are administered before one or more additional therapeutic agents. In some embodiments, the APC 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 a cytokine (such as IL-2), for example, to enhance the persistence of activated immune cells. In some embodiments, the method includes administering a chemotherapeutic agent.

[0408] In some embodiments, the second therapy includes chemotherapy, radiotherapy or immune checkpoint inhibitors. In some embodiments, the second therapy is gene therapy (e.g., gene therapy based on mRNA). In some embodiments, the second therapy includes administering a cancer vaccine (such as a cancer vaccine based on mRNA or a cancer vaccine based on DNA). In some embodiments, the second therapy includes administering an oncolytic virus. In some embodiments, before administering the second therapy, the APC or immune cell is administered. In some embodiments, the APC or immune cell is administered in a neoadjuvant setting.

[0409] In some embodiments, the second therapy comprises treating the patient with radiation. In some embodiments, the radiation site is different from the site of the cancer being treated.

[0410] Thus, for example, in some embodiments, a method of treating an individual having cancer is provided, comprising administering to the individual an effective amount of APCs or immune cells activated by any of the methods described herein, wherein the individual is treated with radiation therapy, and wherein the irradiated site is different from the site of the cancer to be treated.

[0411] In some embodiments, the radiotherapy is selected from: external beam radiotherapy, internal radiotherapy (brachytherapy), intraoperative radiotherapy (IORT), systemic radiotherapy, radioimmunotherapy, and administration of radiosensitizers and radioprotectants. In some embodiments, the radiotherapy is external beam radiotherapy, optionally including three-dimensional conformal radiotherapy (3D-RT), intensity modulated radiotherapy (IMRT), photon beam therapy, image guided radiotherapy (IGRT), and stereotactic radiotherapy (SRT). In some embodiments, the radiotherapy is brachytherapy, optionally including interstitial brachytherapy, intracavitary brachytherapy, intraluminal brachytherapy, and intravenously provided radioactive targeting molecules.

[0412] VI. Compositions containing multiple survival, differentiation and / or maturation factors (S / D / M factors)

[0413] The present application also provides a composition (eg, 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 include those described above.

[0414] In some embodiments, a composition (e.g., a cell culture medium) is provided that comprises a plurality of 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.

[0415] In some embodiments, the IL-10R activator is selected from: IL-10 (e.g., PEGylated IL-10, e.g., PEGylated iloleukin 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 STAT3 downstream of IL-10R (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 present in the culture medium at a concentration of at least about 2 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (eg, about 20 ng / ml).

[0416] In some embodiments, the IFNGR activator is selected from: 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 culture medium at a concentration of at least about 5 ng / ml, optionally at least about 10 ng / ml, further optionally about 10 ng / ml to about 200 ng / ml (e.g., about 50-100 ng / ml).

[0417] 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 culture medium at a concentration of at least about 15 pg / ml, optionally at least about 30 pg / ml, further optionally about 30 pg / ml to about 1 ng / ml (e.g., about 100 pg / ml to about 1 ng / ml).

[0418] 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 culture medium at a concentration of at least about 0.5 ng / ml, optionally at least about 1 ng / ml, further optionally about 0.5 ng / ml to about 30 ng / ml (e.g., about 1-10 ng / ml).

[0419] In some embodiments, the plurality of 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. In some embodiments, the plurality of S / D / M factors include a TNFR activator and an IFNGR activator.

[0420] In some embodiments, the plurality of S / D / M factors includes IL-10, IL-4, TNFα, and IFNγ.

[0421] In some embodiments, the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

[0422] 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 culture medium at a concentration of at least about 30 pg / ml, optionally at least about 50 pg / ml, further optionally 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).

[0423] In some embodiments, the multiple S / D / M factors also include an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from: 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 human IL-6, or human recombinant IL-6. In some embodiments, the IL-6 is present in the culture medium at a concentration of at least about 1 pg / ml, optionally at least about 5 pg / ml, further optionally about 5 pg / ml to about 100 pg / ml (e.g., about 10-50 pg / ml, e.g., about 30 pg / ml).

[0424] In some embodiments, the plurality of S / D / M factors includes IL-10, IL-4, TNFα, IL-6, GM-CSF, and IFNγ.

[0425] In some embodiments, the plurality of maturation factors further comprises one or more of the following group: IL-2, IL-4, IL-17 and M-CSF, agonist antibodies thereof or small molecule activators thereof.

[0426] In some embodiments, a composition (e.g., a cell culture medium) is provided, comprising an IL-10 receptor (IL-10R) activator, optionally the IL-10R activator is IL-10 (e.g., human IL-10, or human recombinant IL-10), further optionally the IL-10 is present in the culture 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., about 10 ng / ml to about 200 ng / ml, e.g., about 20 ng / ml), optionally wherein the culture medium is particularly useful for cancer cells (e.g., monocytes obtained from a cancer patient) or cells (e.g., monocytes) expressing low levels of IL-10R (e.g., at least 20%, 30%, 40%, 50% lower than the expression level of IL-10R in corresponding cells from a reference individual (e.g., a healthy individual)).

[0427] The compositions described herein can be prepared by combining the components into a single composition. In some embodiments, the composition is prepared by culturing immune cells (e.g., T cells, e.g., CD4 T cells, e.g., CD8 T cells) and obtaining a 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.

[0428] In some embodiments, a composition (e.g., cell culture medium) derived from a culture of T cells (e.g., supernatant) treated with anti-CD3 and anti-CD28 antibodies is provided, wherein the culture 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 separated from the PBMCs of the same individual or different individuals, and have not been previously treated with anti-CD3 and / or anti-CD28 antibodies before treatment. In some embodiments, the T cells are separated from the PBMCs of the same individual or different individuals, and have been previously treated with anti-CD3 and / or anti-CD28 antibodies before treatment. In some embodiments, the culture medium is derived from a culture after treating T cells with anti-CD3 and anti-CD28 antibodies for about 1-3 days (optionally 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).

[0429] Exemplary embodiments

[0430] Embodiment 1. A method for stimulating a monocyte population from an individual to produce an antigen presenting cell (APC) population, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, wherein the plurality of S / D / M factors include: 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 γ (IFNγ) receptor (IFNGR) activator.

[0431] Embodiment 2. A method as described in embodiment 1, wherein the IL-10R activator is selected from: IL-10, IL-10 family members, IL-10R agonist antibodies, small molecule activators of IL-10R and activators of STAT3 downstream of IL-10R, optionally wherein the IL-10R downstream STAT3 activator is selected from: IL-10 family cytokines, IL-12 family cytokines, IL-6 family cytokines, small molecule STAT3 activators and G-CSF.

[0432] Embodiment 3. A method as described in embodiment 1, wherein the IL-10R activator is selected from: IL-10, IL-22, IL-19, IL20, IL-24, IL12, IL-23, IL-6, colivirin TFA, gacsenwo 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, colivirin TFA or gacsenwo D.

[0433] Embodiment 4. The method of any one of embodiments 1-3, wherein the plurality of S / D / M factors comprises an IL-4R activator, optionally wherein the IL-4R activator is selected from: IL-4, an IL-4R agonist antibody, and a small molecule activator of IL-4R.

[0434] Embodiment 5. The method of embodiment 4, wherein the IL-4R activator is IL-4.

[0435] Embodiment 6. The method of any one of embodiments 1-5, wherein the plurality of S / D / M factors comprises a TNFR 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.

[0436] Embodiment 7. The method of embodiment 6, wherein the TNFR activator is TNFα.

[0437] Embodiment 8. The method of any one of embodiments 1-7, wherein the plurality of S / D / M factors comprises an IFNGR activator, optionally wherein the IFNGR activator is selected from: IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.

[0438] Embodiment 9. The method of embodiment 8, wherein the IFNGR activator is IFNγ.

[0439] Embodiment 10. The method of any one of embodiments 1-9, wherein the plurality of S / D / M factors are present in a single composition.

[0440] 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 one of the other S / D / M factors in the plurality of S / D / M factors.

[0441] Embodiment 12. The method of any one of embodiments 1-11, wherein 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.

[0442] Embodiment 13. A method as described in embodiment 12, wherein the multiple S / D / M factors include IL-10R activators, TNFα and IFNγ, optionally wherein the multiple 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, optionally wherein the multiple S / D / M factors include IL-10R activators, IL-4, TNFα and IFNγ.

[0443] Embodiment 14. The method of any one of embodiments 1-13, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

[0444] Embodiment 15. The method of embodiment 14, 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.

[0445] Embodiment 16. The method of embodiment 15, wherein the GM-CSFR activator is GM-CSF.

[0446] Embodiment 17. A method as described in any of Embodiments 1-16, wherein the multiple S / D / M factors also include an IL-6 receptor (IL-6R) activator, optionally wherein the IL-6R activator is selected from: IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R.

[0447] Embodiment 18. The method of embodiment 17, wherein the IL-6R activator is IL-6.

[0448] Embodiment 19. The method of any one of embodiments 1-18, wherein the plurality of S / D / M factors are derived from a culture of T cells treated with anti-CD3 antibodies and anti-CD28 antibodies, and optionally the plurality of S / D / M factors are derived from the supernatant of the culture.

[0449] Embodiment 20. A method as described in embodiment 19, wherein the T cells are isolated from PBMCs of the same individual or a different individual, optionally wherein the T cells have not been pre-treated with anti-CD3 antibodies and / or anti-CD28 antibodies prior to treatment.

[0450] Embodiment 21. A method as described in Embodiment 19 or 20, wherein the plurality of S / D / M factors are derived from the culture of the T cells after being treated with anti-CD3 antibody and anti-CD28 antibody for about 1-3 days (optionally about 2 days).

[0451] Embodiment 22. The method of any one of embodiments 1-21, wherein the monocytes are cultured for about 2-3 days in the presence of the S / D / M factor or medium derived from T cell culture.

[0452] Embodiment 23. The method of any one of embodiments 1-22, further comprising contacting the monocyte population with a plurality of refinement factors selected from the group consisting of type I interferons, IFNγ, TNFα, TLR ligands, CD40L or CD40-ligated antibodies, anti-PD-L1 antibodies, and TPI-1, optionally wherein the type I interferons comprise IFNα and / or IFNβ, and optionally wherein the TLR ligand is poly-IC, CpG, or LPS.

[0453] Embodiment 24. A method as described in Embodiment 23, wherein the plurality of optimization factors are provided after contacting the plurality of monocytes with the plurality of S / D / M factors or the culture medium derived from the T cell culture, thereby producing the APC population, and wherein the APC population is cultured in the presence of the plurality of optimization factors for about 1-5 days, optionally wherein the APC population is cultured for about 1 day.

[0454] Embodiment 25. A method as described in embodiment 23 or 24, wherein the multiple optimization factors are provided when a) the monocytes are at least about 50% viable, b) at least about 30% of the APC population displays dendritic cell morphology, and / or c) the APC population 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.

[0455] Embodiment 26. The method of any one of embodiments 23-25, wherein the optimizing factors comprise IFNα, IFNγ, and TNFα.

[0456] Embodiment 27. A method as described in embodiment 26, wherein the optimization factor further comprises at least two agents selected from the group consisting of poly IC, CpG, CD40L, R848 and an anti-PD-L1 antibody, optionally wherein the optimization factor comprises SHP-1 inhibitor Preparations (eg TPI-1) .

[0457] Embodiment 28. A method of promoting survival of a population of monocytes derived from an individual in an in vitro culture, comprising culturing the monocytes in a medium containing one or more molecules that promote expression of an IL-10 receptor (IL-10R) on monocytes.

[0458] Embodiment 29. A method as described in Embodiment 28, wherein the one or more molecules include an IL-10R activator, optionally wherein the IL-10R activator is selected from: IL-10, an IL-10 family member, an IL-10R agonist antibody, a small molecule activator of IL-10R, and an activator of STAT3 downstream of IL-10R, further optionally the IL-10R activator is IL-10.

[0459] Embodiment 30. A method for promoting the survival of a population of monocytes derived from an individual in an in vitro culture, comprising culturing the monocytes in a culture medium containing an IL-10R activator, optionally wherein the IL-10R activator is selected from: IL-10, an IL-10 family member, an IL-10R agonist antibody, a small molecule activator of IL-10R, and an activator of STAT3 downstream of IL-10R, further optionally wherein the IL-10R activator is IL-10.

[0460] Embodiment 31. The method of any one of embodiments 28-30, wherein the monocyte population expresses low levels of IL-10R prior to contacting with the molecule.

[0461] 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: TNFα, a TNFR agonist antibody, and a small molecule activator of TNFR, and optionally wherein the IFNGR activator is selected from: IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR, and further optionally the culture comprises TNFα and / or IFNγ.

[0462] Embodiment 33. A method of increasing the expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual suffering from 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.

[0463] Embodiment 34. A method for promoting survival of a population of monocytes derived from an individual in an in vitro culture, comprising culturing the population of monocytes in a culture medium comprising IL-10, TNFα, and IFNγ.

[0464] Embodiment 35. A method for promoting the differentiation of a monocyte population derived from an individual into antigen presenting cells (APCs) in an in vitro culture, comprising culturing the monocyte population in a culture medium having one or more molecules selected from the following group: an IL-4 receptor (IL-4R) activator, a TNFα receptor (TNFR) activator, and an interferon γ (IFNγ) receptor (IFNGR) activator.

[0465] 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.

[0466] Embodiment 37. The method of any one of embodiments 1-36, wherein the plurality of monocytes are obtained from peripheral blood of the individual, optionally wherein the monocytes express CD 14, and wherein the monocytes are obtained from the peripheral blood.

[0467] Embodiment 38. The method of any one of embodiments 1-37, wherein the individual has cancer.

[0468] Embodiment 39. The method of embodiment 38, wherein the individual has advanced cancer.

[0469] Embodiment 40. The method of any one of embodiments 1-39, wherein the individual has a solid tumor.

[0470] Embodiment 41. The method of any one of Embodiments 1-40, wherein the individual has an inoperable tumor and / or metastasis.

[0471] Embodiment 42. The method of any one of embodiments 1-41, wherein the individual is a human.

[0472] Embodiment 43. A population of APCs produced by the method of any one of embodiments 1-27 and 35-42.

[0473] Embodiment 44. A population of APCs, wherein the APCs a) are MHC-I+ / high, MHC-II+ / high, and CD40+ / high, b) are 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 of CD40 on monocytes, M1 macrophages, M2 macrophages, and / or MoDCs.

[0474] Embodiment 45. A population of APCs, wherein the APCs express higher levels of one or more antigen presenting molecules compared to dendritic cells obtained from healthy humans and cultured with GM-CSF and IL-4 for about 5 days, wherein the antigen presenting molecules are selected from: MHC I, MHC II, CD86, CD80, OX40L, ICAML, ICOSL and CD40, optionally wherein the APCs are generated from monocytes in ex vivo cell culture, further optionally wherein the monocytes are obtained from cancer patients, optionally wherein the APCs express low levels of inhibitory signal molecules, wherein the inhibitory signal molecules are selected from: TGFβR, SIRPα, LIlRBs and Siglec10.

[0475] Embodiment 46. A method for activating an immune cell population, comprising co-culturing the immune cell population with an APC population as described in any one of embodiments 43-45, wherein the APC population is preloaded with one or more neo-antigenic peptides.

[0476] Embodiment 47. The method of Embodiment 46, wherein the method comprises contacting the APC with a composition comprising a plurality of neo-antigenic peptides, and / or the APC has been pre-cultured with the composition.

[0477] Embodiment 48. A method as described in Embodiment 47, wherein the composition comprising a plurality of neoantigenic peptides is a surgical resection of a tumor tissue or a biopsy extract thereof.

[0478] Embodiment 49. A method as described in Embodiment 47, wherein the composition comprising a plurality of neoantigenic peptides is a mixture of tumor cells or an extract thereof isolated from tumor tissue or biopsy.

[0479] Embodiment 50. The method of Embodiment 49, wherein the composition comprising a plurality of neo-antigenic peptides is a mixture of separated neo-antigenic peptides.

[0480] Embodiment 51. A method as described in Embodiment 50, wherein the isolated neoantigenic peptide is a synthetic peptide.

[0481] Embodiment 52. The method of any one of Embodiments 47-51, wherein the APC is allowed to contact with the composition comprising a plurality of neo-antigenic peptides for about 4 hours to about 24 hours.

[0482] Embodiment 53. A method as described in any of embodiments 46-52, wherein the immune cells are selected from: PBMCs, tumor infiltrating T cells (TILs) and T cells, and optionally the immune cells are T cells, optionally the T cells are CD8 T cells and / or CD4 T cells, optionally subjected to at least 3 rounds of activation, wherein each round of co-culture lasts at least about 5 days, wherein at least 2 of the 3 rounds of co-culture do not contain anti-CD3 antibodies or anti-CD28 antibodies.

[0483] Embodiment 54. The method of any one of embodiments 46-53, wherein the co-cultivation is performed for at least 24 hours.

[0484] Embodiment 55. The method of any one of Embodiments 46-54, further comprising expanding the immune cell population after the co-culturing step.

[0485] Embodiment 56. A method as described in Embodiment 55, wherein expanding the immune cell population 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.

[0486] Embodiment 57. The method of any one of Embodiments 46-56, wherein the population of immune cells and the antigen presenting cells are derived from the same individual.

[0487] Embodiment 58. The method of any one of Embodiments 46-57, wherein the population of immune cells and the antigen presenting cells are not derived from the same individual.

[0488] Embodiment 59. An activated immune cell population obtained by the method as described in any one of embodiments 46-58.

[0489] Embodiment 60. A method of treating cancer in a patient, comprising administering to the patient the APC population of embodiments 43-45 and / or the activated immune cells of embodiment 59.

[0490] Embodiment 61. The method of embodiment 60, wherein the APCs or activated immune cells are administered intratumorally, intraperitoneally or intravenously.

[0491] 62. The method of embodiment 61, wherein the activated immune cells are administered at a dose of about 10 7 -10 9 Cell administration.

[0492] 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.

[0493] Embodiment 64. The method of embodiment 63, wherein the method comprises treating the patient with radiation.

[0494] Embodiment 65. The method of Embodiment 64, wherein the irradiation site is different from the site of the cancer to be treated.

[0495] Embodiment 66. The method of any one of Embodiments 60-65, wherein the APCs or activated immune cells administered to the patient are derived from the patient.

[0496] 67. The method of any one of embodiments 60-65, wherein the APCs or activated immune cells administered to the patient are not derived from the patient.

[0497] Embodiment 68. The method of any one of embodiments 60-67, wherein the cancer to be treated is a solid tumor.

[0498] Embodiment 69. A composition comprising a plurality of survival, differentiation and / or maturation factors (S / D / M factors), wherein the plurality of S / D / M factors include: 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.

[0499] Embodiment 70. The composition of embodiment 69, 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 STAT3 downstream of IL-10R.

[0500] Embodiment 71. A composition as described in embodiment 70, wherein the IL-10R activator is IL-10, IL-22, IL-19, IL-20, IL-24, IL-12, IL-23, clovaline TFA or glucagon D, optionally the IL-10R activator is IL-10.

[0501] Embodiment 72. The composition of any one of Embodiments 69-71, wherein the plurality of S / D / M factors comprises an IL-4R activator, optionally wherein the IL-4R activator is selected from: IL-4, an IL-4R agonist antibody, and a small molecule activator of IL-4R.

[0502] Embodiment 73. The composition of embodiment 72, wherein the IL-4R activator is IL-4.

[0503] Embodiment 74. The composition of any one of embodiments 69-73, wherein the plurality of S / D / M factors comprises a TNFR 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.

[0504] Embodiment 75. The composition of embodiment 74, wherein the TNFR activator is TNFα.

[0505] Embodiment 76. The composition of any one of Embodiments 69-75, wherein the plurality of S / D / M factors comprises an IFNGR activator, optionally wherein the IFNGR activator is selected from: IFNγ, an IFNGR agonist antibody, and a small molecule activator of IFNGR.

[0506] Embodiment 77. The composition of embodiment 76, wherein the TNFR activator is IFNγ.

[0507] Embodiment 78. The composition of any one of embodiments 69-77, wherein 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.

[0508] Embodiment 79. The composition of embodiment 78, wherein the plurality of S / D / M factors comprises IL-10, IL-4, TNFα, and IFNγ.

[0509] 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.

[0510] 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.

[0511] Embodiment 82. The composition of embodiment 81, wherein the GM-CSFR activator is GM-CSF.

[0512] Embodiment 83. A composition as described in any of Embodiments 69-82, 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: IL-6, an IL-6R agonist antibody, and a small molecule activator of IL-6R.

[0513] Embodiment 84. The composition of embodiment 83, wherein the IL-6R activator is IL-6. Example

[0514] The following examples are intended to be merely exemplary of the present invention and therefore should not be considered to limit the present invention in any way.The following examples and detailed description are provided by way of illustration and not limitation.

[0515] Example 1

[0516] Experiments described herein investigating peripheral monocytes from cancer patients with advanced malignancies (e.g., stage II, III, or IV) and inoperable tumors and / or metastases found that these monocytes (referred to as cancer monocytes or cMo) showed a different response to the classical macrophage / DC-differentiation factors M-CSF and / or GM-CSF than monocytes from healthy donors (healthy monocytes or Mo). Figures 1A-1C ), Mo were easily driven by M-CSF and differentiated into macrophages within 4-5 days, whereas cMo showed non-responsiveness (no cell attachment or morphological changes) under the same treatment, followed by cell death within 2 days. Similarly, cMo responded poorly to GM-CSF alone or GM-CSF plus IL-4, which drove DC differentiation (data not shown). More than 20 cMo samples from cancer patients with various solid tumors (e.g., lung cancer, kidney cancer, liver cancer, colorectal cancer, thymic cancer, and sarcoma) have been tested, and in all cases, cMo consistently showed non-responsiveness or poor response to M-CSF and GM-CSF, but with a high rate of cell death ( Figure 4 ). Parallel experiments testing the same M-CSF and GM-CSF reagents successfully differentiated Mo from healthy donors with >90% viability.

[0517] Further studies on M-CSF- and GM-CSF-mediated signal transduction (eg Figure 2A ) demonstrated 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, respectively, compared to total Akt and Erk1 / 2 protein levels); however, this signaling event was not replicated in cMo. In contrast, in response to M-CSF and GM-CSF, cMo displayed enhanced apoptotic signaling, as well as increased cleavage of caspase-9 and caspase-3, leading to cell death ( Figure 2B Examination of Akt and Erk1 / 2, as well as other signaling molecules, detected similar total levels of Mo relative to cMo ( Figure 2C ), excluding the lack of protein phosphorylation due to decreased protein translation.

[0518] Examination of cell surface cytokine receptors revealed that cMo expressed reduced levels of MCSF-R and GMCSF-R compared with Mo ( Figure 1D ), which partially explains the poorer response of cMo to these differentiation factors, suggesting that insufficient stimulation intensity not only makes cell survival signaling impossible, but also triggers 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 their results are consistent with the data presented herein. Previous transcriptional profiling by different groups has also confirmed different gene expression patterns between cMo (or TEMo) and Mo from healthy donors (Bergenfelz et al., 2015; Cassetta et al., 2019; Chittezhath et al., 2014; Ramos et al., 2020).

[0519] Example 2

[0520] Although cMo differentiation failed with M-CSF and GM-CSF, conditioned medium from T cells activated by TCR ligation was found to induce cMo differentiation into phagocytic APCs.

[0521] In these experiments (see Figure 3A ), freshly isolated T cells from PBMCs of healthy donors or autologous cancer patients were linked to anti-CD3 and anti-CD28 antibodies to trigger 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 co-stimulation ( Figures 3A-3D). After 2 days, cMo were treated with the T cell culture medium. In all experiments using cMo from each cancer patient ( Figure 4 ), medium from TCR-activated T cells rapidly induced cMo attachment to the dish (1-4 hours). IL-10, along with high levels of other cytokines (e.g., IFNγ, TNFα, IL-4, GM-CSF, and IL-6) produced by the same CD4 T cells, was found to support cMo survival and differentiation into a unique type of APC (termed κAPC), as indicated by changes in cell morphology ( Figure 3E-3G In addition, this culture condition induces their phenotype to differentiate into professional APCs ( Fig. 3I Within 2 days, cMo-differentiated APCs displayed increased expression of cell surface antigen presentation machinery, including high levels of MHC-I, MHC-II, co-stimulatory molecules CD80 and CD86, and APC activation molecules CD40 and PD-L1 ( Figure 3E , high expression of MHC-II that marks differentiation into professional APCs).

[0522] Taken together, these results suggest that certain factors produced by TCR-stimulated T cells not only support cMo survival (attachment) but also drive cMo differentiation into professional APCs. The culture medium of TCR-stimulated T cells harvested on day 2 is called Kanelian X1 (or KX1).

[0523] Example 3

[0524] Further investigation of Carnelian X1 revealed that biological components that support cMo survival and differentiation are present in fractions with a molecular weight (MW) of 30-100 KD, which are enriched with cytokines produced by T cells while excluding exosomes and lipid vesicles. Cytokine analytes identified IFNγ, IL-2, IL-10, TNFα, GM-CSF, IL-6, IL-17, M-CSF, and IL-4 ( Figure 5A Specifically, high levels of IL-10 and IL-6 were detected in Carnelian X1. Additional cytokine depletion analysis confirmed the critical role of IL-10 and its ability to significantly reduce cMo attachment and survival (60-80%). Figure 5B Adding IL-10 (recombinant form) back to IL-10-depleted Carnelian X1 restored its viability in a dose-dependent manner ( Figure 5C Inhibition of IL-10R-associated JAKs by JAK inhibitor I, or inhibition of downstream STAT3 by inhibitors C188-9 and napacasin, abolished the effect of KX1 in a dose-dependent manner ( Figure 5D), leading to cMo cell apoptosis. In contrast, supplementation of the STAT3 activator cleaverine TFA or galactopyranoside D to IL-10-depleted KX1 medium (KX1 low IL-10) restored cMo survival and promoted cMo differentiation into κAPC ( Figure 5E Studies on other cytokines that activate STAT3 have found that IL-10 family members (e.g., IL-19, IL-20, IL-22, and IL-24) and IL-12 family members (IL-12 and IL-23) can replace IL-10 to support cMo survival and differentiation ( Fig. 5F and Figure 5G IL-12 mechanism involves the induction of IL-10 ( Figure 5H ); In addition, IL-12 is known to directly activate STAT3. Although IL-6 family cytokines activate STAT3 through the receptor gp130, treatment of cMo with IL-6 or its family member IL-11 only modestly supports cMo survival and differentiation ( Figure 5J ). KX1 medium depleted of G-CSF and IL-10 (KX1 低IL-10 ) treatment showed that the cMo survival level was similar to that of IL-6 treatment ( Figure 5K ). However, IL-10 appears to only weakly affect cMo differentiation into APC ( Fig. 6E ), because the residual cells (5-20%) that survive IL-10 depletion continue to differentiate to adopt a DC-like morphology and increased expression of MHC-II and antigen presentation machinery.

[0525] IL-10 / IL-10R signal transduction activates the downstream STAT3 signal transduction pathway. Other cytokines of the IL-10 family can activate STAT3, and further show similar levels of induction of cMo survival in vitro. Similarly, IL-12 and IL-23 are STAT3 activators (e.g., produced by activating IL-10 and signal transduction in cMo), and the two cytokines show that cMo survival increases at a ratio higher than IL-6 but lower than IL-10. IL-6 is a known activator of the STAT3 pathway, however, when cultured with IL-6 alone, cMo survival is the lowest, thereby highlighting the unexpected results of IL-10, IL-12 and its cytokine family members as the pro-survival mechanism of cMo in vitro. Small molecules that activate STAT3 show similar pro-survival effects as IL-10. See Table 1 below.

[0526] Table 1. STAT3 activators that promote cMo survival in vitro.

[0527]

[0528] Three other cytokines, TNFα, IFNγ, and IL-4, were also found to be important. Depletion of TNFα or IFNγ in carnelian X1 reduced cMo survival (approximately 20%) and differentiation of surviving cMo into APCs (30 to 70% reduction in MHC-II) ( Figure 5B On the other hand, IL-4 did not affect cMo survival / attachment, but significantly affected cMo differentiation into APCs (about 50% reduction, Fig. 6A ). We tested the differentiation of cMo from >100 cancer patient samples. These patients had...

Claims

1. A method for stimulating a monocyte population from an individual to produce an antigen presenting cell (APC) population, comprising contacting the monocyte population with a plurality of survival, differentiation and / or maturation factors (S / D / M factors) separately or simultaneously, thereby obtaining an APC population, The multiple S / D / M factors include: 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.

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

3. The method of claim 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, colivirin TFA, gadolinium 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, colivirin TFA or gadolinium D.

4. The method of any one of claims 1-3, wherein the plurality of S / D / M factors comprises an IL-4R activator, optionally wherein 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 of claim 4, wherein the IL-4R activator is IL-4.

6. The method of any one of claims 1-5, wherein the plurality of S / D / M factors comprises a TNFR 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.

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

8. The method of any one of claims 1-7, wherein the plurality of S / D / M factors comprises an IFNGR activator, optionally wherein 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 of claim 8, wherein the IFNGR activator is IFNγ.

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

11. The method of any one of claims 1-10, wherein 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.

12. The method of claim 11, 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.

13. The method of any one of claims 1-12, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

14. The method of claim 13, 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.

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

16. The method of any one of claims 1-15, 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.

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

18. The method of any one of claims 1-17, further comprising contacting the monocyte population with a plurality of optimizing factors selected from the group consisting of type I interferons, IFNγ, TNFα, TLR ligands, CD40L or CD40-ligated antibodies, anti-PD-L1 antibodies, and TPI-1, optionally wherein the type I interferons include IFNα and / or IFNβ, and optionally wherein the TLR ligand is poly-IC, CpG, or LPS.

19. The method of claim 18, wherein the optimizing factors include IFNα, IFNγ, and TNFα.

20. The method of claim 19, wherein the optimizing factor further comprises at least two agents selected from the group consisting of poly IC, CpG, CD40L, R848, and anti-PD-L1 antibodies, optionally wherein the optimizing factor comprises a SHP-1 inhibitor (e.g., TPI-1).

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

22. A method for promoting the survival of a monocyte population derived from an individual in an in vitro culture, comprising culturing the monocyte population in a culture medium containing 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 agonist antibody, a small molecule activator of IL-10R, and an activator of STAT3 downstream of IL-10R, and further optionally wherein the IL-10R activator is IL-10.

23. A method of increasing the expression of IL-10 receptor (IL-10R) in a population of monocytes from an individual suffering from 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.

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

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

26. The method of any one of claims 1-23, wherein the individual has cancer.

27. A population of APCs produced by the method of any one of claims 1-20 and 23-24.

28. A population of APCs, wherein the APCs: a) are MHC-I+ / high, MHC-II+ / high, and CD40+ / high, b) are 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 of CD40 on monocytes, M1 macrophages, M2 macrophages, and / or MoDCs.

29. A method for activating an immune cell population, comprising co-culturing the immune cell population with the APC population of claim 25 or 26, wherein the APC population is preloaded with one or more neo-antigenic peptides.

30. The method of claim 27, wherein the method comprises contacting the population of APCs with a composition comprising a plurality of neo-antigenic peptides, and / or the population of APCs has been pre-incubated with the composition.

31. The method of claim 27 or 28, wherein the immune cells are selected from: PBMCs, tumor infiltrating T cells (TILs) and T cells, optionally the immune cells are T cells, optionally the T cells are CD8 T cells and / or CD4 T cells.

32. The method of claim 29, wherein the activation is performed for at least 3 rounds, wherein each round of co-culture lasts for at least about 5 days, wherein 2 of the 3 rounds of co-culture do not comprise anti-CD3 antibody or anti-CD28 antibody.

33. The method of any one of claims 27-30, wherein the immune cell population and the antigen presenting cells are derived from the same individual.

34. The method of any one of claims 27-30, wherein the immune cell population and the antigen presenting cells are not derived from the same individual.

35. An activated immune cell population obtained by the method of any one of claims 27-32.

36. A method of treating cancer in a patient, comprising administering to the patient the APC population of claim 25 or 26 and / or the activated immune cells of claim 33.

37. 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.

38. The composition of claim 34, 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 STAT3 downstream of IL-10R.

39. The composition of claim 35, wherein the IL-10R activator is IL-10, IL-22, IL-19, IL20, IL-24, IL12, IL-23, IL-6, Cliverin TFA or Garvena D, optionally wherein the IL-10R activator is IL-10.

40. The composition of claim 35 or 36, wherein 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.

41. The composition of claim 37, wherein the plurality of S / D / M factors comprises IL-10, IL-4, TNFα, and IFNγ.

42. The composition of any one of claims 35-38, wherein the plurality of S / D / M factors further comprises a GM-CSF receptor (GM-CSFR) activator.

Images