IL323635A - Methods for enhancing the immunogenicity of cellular vaccines - Google Patents

Description

METHODS FOR ENHANCING THE IMMUNOGENICITY OF CELLULAR VACCINES BACKGROUND [0001] Autologous dendritic cell (DC) vaccines with tumor antigens have been used clinically with limited therapeutic effects. Semi-allogeneic DC-based immunotherapy is still controversial, but it can be an alternative source and more attractive than autologous DC vaccines because the "off-the-shelf" DCs can be used for multiple patients without lengthy individual manufacturing time and may provide additional "allogeneic help". The present disclosure satisfies these needs and provides related advantages as well.

BRIEF SUMMARY [0002] In one aspect, the present disclosure provides an engineered mammalian dendritic cell comprising one or more exogenous alleles of at least one Major Histocompatibility Complex (MHC) class II gene. id="p-3"

[0003] In some embodiments, the one or more exogenous alleles are introduced through homologous recombination or through transfection or transduction of one or more expression vectors into the cell. id="p-4"

[0004] In some instances, the exogenous alleles comprise an exogenous allele of a first MHC class II gene and an exogenous allele of a second MHC class II gene. In other instances, the exogenous alleles comprise a first exogenous allele of an MHC class II gene and a second exogenous allele of the same MHC class II gene. id="p-5"

[0005] In some embodiments, the engineered mammalian dendritic cell is an engineered human dendritic cell. In some embodiments, the MHC class II gene comprises an HLA class II alpha subunit gene, an HLA class II beta subunit gene, or a combination thereof. In some embodiments, the MHC class II gene comprises an HLA-DR gene, an HLA-DP gene, an HLA-DQ gene, an HLA-DM gene, an HLA-DO gene, or a combination thereof. In some embodiments, the HLA-DR gene comprises an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, an HLA-DRB5 gene, or a combination thereof. In some embodiments, the HLA-DP gene comprises an HLA-DPA1 gene, an HLA-DPB1 gene, or a combination thereof. In some embodiments, the HLA-DQ gene comprises an HLA-DQA1 gene, an HLA-DQB1 gene, or a combination thereof. id="p-6"

[0006] In some embodiments, the engineered mammalian dendritic cell comprises an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof. id="p-7"

[0007] In some instances, the cell is engineered from a cell line. In some embodiments, the cell line is HL-60, THP-1, K562, MUTZ3, or an immortalized dendritic cell. In some embodiments, the immortalized dendritic cell expresses HTLV-1 transactivator (Tax) protein, SV40 proteins, and/or hTERT. id="p-8"

[0008] In other instances, the cell is engineered from a primary cell. In some embodiments, the primary cell is from a patient. In some embodiments, the patient has a cancer. id="p-9"

[0009] In another aspect, the present disclosure provides a composition comprising an engineered mammalian dendritic cell comprising one or more exogenous alleles of at least one Major Histocompatibility Complex (MHC) class II gene. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant. id="p-10"

[0010] In a further aspect, the present disclosure provides a method for semi-allogeneic dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, prior to the administering, the method further comprises: (i) obtaining an MHC class II allele profile by genotyping a plurality of MHC class II genes in a biological sample from the subject; and (ii) selecting an engineered mammalian dendritic cell for administering to the subject, wherein the engineered mammalian dendritic cell comprises one or more mismatches to the MHC class II allele profile of the subject. In some embodiments, the method further comprises administering to the subject a regulatory T cell inhibitory agent (Treg agent). In some embodiments, the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof. In some embodiments, the Treg agent is administered after administering the pharmaceutical composition. In some embodiments, the subject is a human. In some embodiments, the human has a cancer, wherein the engineered dendritic cell comprises a tumor-specific antigen or a fragment thereof. id="p-11"

[0011] In another aspect, the present disclosure provides a method for autologous dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein, wherein the engineered mammalian dendritic cell is from a primary immune cell of the subject. In some embodiments, prior to the administering, the method further comprises: (i) obtaining the primary immune cell or a plurality thereof from the subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cell to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cell into an engineered mammalian dendritic cell by: (a)introducing into the primary immune cell one or more exogenous MHC class II alleles comprising at least one mismatch to the endogenous MHC class II allele profile of the subject; and (b) introducing into the primary immune cell an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof. In some embodiments, (iii) further comprises: (c) incubating the primary immune cell with Lipopolysaccharide (LPS), interferon-gamma (IFN-γ), or a combination thereof. In some embodiments, the method further comprises administering to the subject a Treg agent. In some embodiments, the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof. In some embodiments, the Treg agent is administered after administering the pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS id="p-12"

[0012] FIG. 1 depicts the three mouse strains used in the study. (A) C57BL/6J mouse is wild type (WT) with MHC haplotype H2b; (B) B6.C-H2-Kbm1/ByJ (bm1) mouse is MHC class I mutant with haplotype H2bm1. The B6.C-H2-Kbm1/ByJ mice differ from the C57BL/6J Kb allele by nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 152, 155, and 156 of the α2 domain. (C) B6(C)-H2-Ab1bm12/KhEgJ (bm12) mouse is MHC class II mutant with haplotype H2bm12. The B6(C)-H2-Ab1bm12/KhEgJ mice differ from the C57BL/6J IAb allele by 3 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 67, 70, and 71 of the β1 domain. id="p-13"

[0013] FIG. 2 depicts schematic drawings of antigen presenting cells (APCs) with MHC alloantigens H2-Db, H2-Kb, and H2-IAb and the genetic loci of the murine H-2 complex. The Epeptide is a peptide from human papilloma virus (HPV) protein E7, and only binds to MHC class I, H-2Db. (A) APC WT contains wild type MHC alloantigens H2-Db, H2-Kb, and H2-IAb, and H2-Db presents the E7 peptide. (B) APC mutant bm1 contains wild type H2-Db which presents the Epeptide, and wild type H2-IAb, and mutant H2-Kbm1 which stimulates MHC class I allogeneic help. (C) APC mutant bm12 contains wild type H2-Db which presents the E7 peptide, and wild type H2-Kb, and mutant H2-IAbm12 which stimulates MHC class II allogeneic help. (D) indicates the genetic loci of the murine H-2 complex, also known as the murine major histocompatibility complex (MHC). Classical MHC class I comprises H-2D, H-2K and H-2L subclasses in the K region and D region, non-classical MHC class Ib comprises H-2Q, H-2M and H-2T subclasses in the Q/T/M region. Classical MHC class II comprises H-2A(IA) and H-2E(IE) and non-classical MHC-IIb comprises H-2P (P), H-2M (DM) and H-2O (DO) subclasses. All MHC class II subclasses are located in the I region. MHC class III is located in the S region. id="p-14"

[0014] FIG. 3 depicts the expression of MHC class II (IAIE), CD11c, CD40, CD80 and CDin immature and mature BMDCs as measured by flow cytometry. Dendritic cells (DC) were generated from bone marrow (BM) progenitors of WT C57BL/6 mice and cultured with 20 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7-, 8-, 9-, 10-, and 12-days before being harvested. The cells were incubated with or without 100 ng/ml lipopolysaccharide (LPS, Sigma-aldrich, Heidelberg, Germany) for overnight, and both matured and immature BMDCs were measured by flow cytometry. (A) indicates CD11c+ IAIE+ cells were over 80% after 8 days culturing and reached over 90% after 10 days culturing with GM-CSF. (B-E) indicate the maturation of BMDCs not only stimulate the expression of CD86, also increase the expression of MHC class II (IAIE), CD40, and CD80. id="p-15"

[0015] FIG. 4 depicts the maturation of BMDCs. BMDCs from C57BL/6J (WT), B6.C-H2-Kbm1/ByJ (bm1), or B6(C)-H2-Ab1bm12/KhEgJ (bm12) after 10 days culture with GM-CSF were pulsed with E743-77 (10 µg/ml) for 1-2 hours and maturated with LPS (100 ng/ml) overnight. The BMDCs were tested for CD11c, H2-Db, CD40, CD80 and CD86 expression by flow cytometry. (A) Over 90% of BMDCs from all three mouse strains (WT, bm1 and bm12) were CD11c+, indicating the majority of BMDCs were mature. The expression of H2-Db (B) and CD40, CDand CD86 (C) were also examined by flow cytometry, also demonstrating mature phenotype. id="p-16"

[0016] FIG. 5 depicts tumor volume growth curves of the mice in the first mouse study. Female C57BL/6J mice (13 weeks old) were inoculated subcutaneously with 1 x 10 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter. On Day 8, 13, 19, 23 and 28 after the TC-1 cell inoculation, mice were vaccinated intradermally with 2 x 10 (1.6 x 10on Day 19) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ (E7-mBMDC bm1), or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). (A) depicts tumor volume growth curves of the mice in 4 groups. Data are shown as Mean ± SEM (n = 5 or 6 as indicated in the legend). Compared to the PBS control group, vaccination with the E7-BMDC WT slowed tumor growth. The E7-BMDC bm1 (MHC class I mutant) showed a similar effect as the E7-BMDC WT. The E7-BMDC bm12 (MHC class II mutant) showed a greater effect in limiting tumor growth than either the E7-BMDCs WT or the E7-BMDC bm1 (MHC class I mutant). (B) depicts tumor volume growth curves of individual mice. The tumor on one mouse had disappeared after the fifth vaccination with the E7-BMDC bm12 (MHC class II mutant). id="p-17"

[0017] FIG. 6 depicts tumor volume growth of the mice on different days in the first mouse study. (A) depicts tumor volume growth on day 19; (B) depicts tumor volume growth on day 21; (C) depicts tumor volume growth on day 34; (D) depicts tumor weight on day 35. The PBS depicts the control group of the mice injected with PBS; E7-mBMDC WT depicts the mice vaccinated with syngeneic E7-pulsed BMDCs from C57BL/6J; E7-mBMDC bm1 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ; and E7-mBMDC bm12 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ. The statistically significant differences in tumor volume (A-C) or tumor weight (D) between groups were analyzed by the Mann Whitney test and indicated as * < P= 0.05, ** < P= 0.01. id="p-18"

[0018] FIG. 7 depicts tumor volume growth of the mice in the second mouse study. Female C57BL/6J mice (9 weeks old) were inoculated subcutaneously with 1 x 10 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm long diameter. On Day 9, 14, 19, and 24 after the TC-1 cell inoculation, mice were vaccinated intradermally 2 x 10 (1.6 x 10 on Day 14) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ (E7-mBMDC bm1), or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). (A) depicts tumor volume growth curves of the mice in groups. Data are shown as Mean ± SEM (n = 5 or 6 as indicated in the legend). Compared to the PBS control group, vaccination with the E7-BMDC WT slowed tumor growth. The E7-BMDC bm1 (MHC class I mutant) showed a similar effect as the E7-BMDC WT. The E7-BMDC bm(MHC class II mutant) showed a greater effect than either the E7-BMDCs WT or the E7-BMDC bm1 (MHC class I mutant). (B) depicts the ratio of tumor/body weight of the mice on Day 28 after TC-1 cell inoculation. (C) depicts the body weight of the mice on Day 28 after TC-1 cell inoculation. (D) depicts the tumor weight of the mice on Day 28 after TC-1 cell inoculation. PBS depicts the control group of the mice injected with PBS; E7-mBMDC WT depicts the mice vaccinated with syngeneic E7-pulsed BMDCs from C57BL/6J; E7-mBMDC bm1 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ; and E7-mBMDC bm12 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ. The statistically significant differences between groups were analyzed by the Mann Whitney test and indicated as * < P= 0.05, ** < P= 0.01. id="p-19"

[0019] FIG. 8 depicts tumor volume growth curves of mice in a CD4 or CD8 T cell depletion study. Female C57BL/6J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 TC-cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter. Intraperitoneal injection of anti-CD4 antibody at an early stage of tumor growth (Early aCD4), or anti-CD8 antibody (aCD8), was started on day 6 (200 µg/mouse) after the inoculation and continued every 2-4 days (100 µg/mouse) to the end of the experiment. Intraperitoneal injection of anti-CD4 antibody at a late stage of tumor growth (late aCD4) was started on day 17 (2µg/mouse) after the inoculation and continued every 2-4 days (100 µg/mouse) to the end of the experiment. On Day 7, 12, 17, 22 and 27 after the TC-1 cell inoculation, mice were vaccinated intradermally with 2 x 10 syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT) or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). (A) depicts tumor volume growth curves of the mice in 3 groups with isotype control antibody (Isotype). Data are shown as Mean ± SEM (n = 4 or 5). Compared to the PBS control group, vaccination with the E7-BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT) slowed tumor growth, but the E7-BMDC bm12 (MHC class II mutant) with isotype control antibody (Isotype + the E7-BMDC bm12) showed a greater effect on limiting tumor growth than the E7-BMDCs WT with isotype control antibody (Isotype + the E7-BMDC WT). (B) depicts tumor volume growth curves of the mice in 4 groups either injected with phosphate-buffered saline (PBS) or vaccinated with the E7-BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT), anti-CD4 antibody at an early stage of tumor growth (Early aCD4 + the E7-BMDC WT), or anti-CD8 antibody (aCD+ the E7-BMDC WT). The groups vaccinated with E7-BMDC WT with isotype control antibody or anti-CD4 antibody showed similar growth curves. (C) depicts tumor volume growth curves of the mice in 5 groups either injected with phosphate-buffered saline (PBS) or vaccinated with the E7-BMDC bm12 with isotype control antibody (Isotype + E7-BMDC bm12), anti-CD4 antibody at an early stage of tumor growth (Early aCD4 + E7-BMDC bm12), anti-CD4 antibody at a late stage of tumor growth (late aCD4 + E7-BMDC bm12), or anti-CD8 antibody (aCD8 + E7-BMDC bm12). The group vaccinated with E7-BMDC bm12 with isotype control antibody (Isotype + E7-mBMDC bm12) showed a greater effect on limiting tumor growth. Administration of anti-CDantibody to the vaccinated mice at an early stage of tumor growth (Early aCD4 + E7-mBMDC bm12) inhibited such effect until day 17. Administration of anti-CD4 antibody to the vaccinated mice at a late stage of tumor growth (late aCD4 + E7-mBMDC bm12) showed the best effect on suppressing tumor growth after the 5th vaccination dose. (C) shows that CD4 depletion at a late stage of tumor growth had a greater effect on limiting tumor growth in mice vaccinated with E7-BMDC bm12. id="p-20"

[0020] FIG. 9 depicts tumor volume growth of the mice on different days of the CD4 or CD8 T cell depletion study shown in FIG. 8 . (A) depicts tumor volume growth on day 14; (B) depicts tumor volume growth on day 20; (C) depicts tumor volume growth on day 29. These data show that early CD4 depletion reduced the vaccine effect in the "early aCD4 + E7-mBMDC bm12" mice (FIG. 9A); however, late CD4 depletion enhanced the vaccine effect in the "late aCD4 + E7-mBMDC bm12" mice on limiting late-stage tumor growth (FIG. 9C), indicating that allo-CD4+ Th response occurred at an early stage of tumor growth and CD4 depletion covered Treg depletion at an early stage of tumor growth. The statistically significant differences in tumor volume (A-C) between groups were analyzed by the Mann Whitney test and indicated as * < P= 0.05. id="p-21"

[0021] FIG. 10 depicts tumor volume growth of the mice in a Treg cell depletion study. Six groups of female B6.129(Cg)-Foxp3tm3(Hbegf/GFP)Ayr/J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 TC-1 cells. Seven days after the inoculation, all tumors on the mice reached around 5 mm in diameter. The first group of mice was injected with phosphate-buffered saline (PBS) only as a control group. Two groups of mice (2 doses of E7-mBMDC bm12) were vaccinated intradermally 2 x 10 semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12) on day 8 and 13 after the TC-1 cell inoculation. Three groups of mice (5 doses of E7-mBMDC bm12) received the same vaccine on day 8, 13, 18, 23, and 28. These Foxp3DTR knock-in mice express the human diphtheria toxin (DT) receptor, and they are depleted for Treg cells when they are injected with DT. Among the two "2 doses of E7-mBMDC bm12" groups, one group of mice (late DT + 2 doses of E7-mBMDC bm12) received late DT injection in which intraperitoneal injection of 10 µg/kg DT was started on day 15 after the inoculation, and the other group did not receive DT treatment (2 doses of E7-mBMDC bm12). Among the three "5 does of E7-mBMDC bm12" groups, one group of mice (Early DT + 5 doses of E7-mBMDC bm12) received early DT injection in which intraperitoneal injection of 10 µg/kg DT was started on day 6 after the inoculation; one group of mice (late DT + 5 doses of E7-mBMDC bm12) received late DT injection started on day 15 after the inoculation; and the last group did not receive DT treatment (5 doses of E7-mBMDC bm12). All the mice treated with DT continued receiving DT treatment every 2-4 days to the end of the experiment. Data are shown as Mean ± SEM (n = 4 or 5). The statistically significant differences between groups were analyzed by the Two-way ANOVA test with the post hoc Tukey’s multiple comparison test and indicated as * < P= 0.05. id="p-22"

[0022] FIG. 11 depicts IFNγ production in TC-1 bearing mouse CD4 or CD8 T cells co-cultured with mBMDC WT, E7-mBMDCs WT, mBMDC bm12, or E7-mBMDC bm12. CD4 and CD8 T cells were isolated from spleens of TC-1 bearing mice 8 days after TC-1 inoculation and incubated at a concentration of 2 x 10 cells per well, alone or co-cultured with BMDCs (1 x 10 cells per well, with or without E7-pulsing), in a 96-well round plate for 24 hrs or 72 hrs. The isolated CDT cells, either alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDCs WT (E7-WT CD8), mBMDC bm12 (bm12 CD8), or E7-pulsed mBMDC bm12 (E7-bm12 CD8), were tested for IFNγ production via intracellular staining and flow cytometry (A) and for IFNγ production in the supernatant by ELISA (C). The isolated CD4 T cells, either alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDCs WT (E7-WT CD4), mBMDC bm(bm12 CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD4), were tested for IFNγ production via intracellular staining and flow cytometry (B) and for IFNγ production in the supernatant by ELISA (C). A mix of 2 x 10 CD8 and 2 x 10 CD4 T cells, either alone (CD8+ CD4) or co-cultured with mBMDC WT (WT CD8+CD4), E7-pulsed mBMDCs WT (E7-WT CD8+CD4), mBMDC bm(bm12 CD8+CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD8+CD4), were also tested (A-C). (A) depicts IFNγ production of CD8 T cells via intracellular staining and flow cytometry 24 hrs after co-culture. CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8) generated significantly higher levels of IFNγ in comparison to a mix of CD8 and CD4 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8+CD4). CD8 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8) and a mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bm(E7-bm12 CD8+CD4) showed similar high IFNγ production compared to CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8). These data indicate that allo-CD4+ Th response from E7-mBMDC bm12 vaccination can stimulate CD8 cells to produce a high level of IFNγ. (B) depicts IFNγ production of CD4 T cells by via intracellular staining and flow cytometry 24hrs after co-culture. CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) generated significantly higher levels of IFNγ in comparison to CD4 T cells co-cultured with E7-mBMDC WT (E7-WT CD4). (C) depicts IFNγ production in the supernatant of CD4 and/or CD8 T cell cultures by ELISA 72hrs after co-culture. While no IFNγ was detected in most samples, CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD4) showed a good amount of IFNγ release, significantly higher than CD8 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8). Robust IFNγ production was observed in the supernatant of the mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4). The statistically significant differences between groups were analyzed by one-way ANOVA test and indicated as * < P= 0.05, ** < P= 0.01, *** < P = 0.005, **** < P= 0.0001.

DETAILED DESCRIPTION I. Introduction id="p-23"

[0023] The present disclosure is based, in part, on the inventors’ discovery that engineered mammalian dendritic cells (DCs) comprising semi-allogeneic Major Histocompatibility Complex (MHC) class II alleles can elicit a robust immune response in a subject. In particular, the present disclosure relates to engineering mammalian DCs to express one or more exogenous MHC class II alleles. The engineered mammalian DCs can be derived from a cell line and used as an "off-the-shelf" semi-allogeneic cellular vaccine. The engineered mammalian DCs can be derived from a subject and returned to the subject after modification for cancer and/or disease treatment as an autologous cellular immunotherapy. The engineered mammalian DCs can be derived from one subject and used to treat other subject as a semi-allogeneic cellular immunotherapy. id="p-24"

[0024] The inventors discovered that semi-allogeneic murine bone marrow dendritic cells (BMDCs) expressing one or more exogenous MHC class II alleles are more effective in suppressing tumor growth in mice than syngeneic DC-based cancer vaccines or semi-allogeneic BMDCs expressing one or more exogenous MHC class I alleles. DCs are antigen-presenting cells expressing both MHC class I and class II molecules. MHC class I molecules activate CD8+ T cells (killer T cells) to kill targeted cells, and MHC class II molecules activate CD4+ T cells (helper T cells) to help other immune cell activities. Without being bound by any theory, the expression of at least one exogenous allele of an MHC class II molecule provides additional help to the DC by enhancing the potency of antigen presentation and eliciting an allo-CD4+ Th response, therefore producing a more potent anti-cancer response. The engineered mammalian DCs described herein can be used as either a semi-allogeneic or autologous DC-based vaccine. id="p-25"

[0025] Described herein are compositions of engineered mammalian DCs expressing one or more exogenous MHC class II alleles and methods of using these cells for semi-allogeneic or autologous cellular immunotherapy of diseases such as cancer. Additionally, pharmaceutical compositions and kits containing the "off-the-shelf" engineered mammalian DCs are also provided.

II. Definitions id="p-26"

[0026] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined. id="p-27"

[0027] The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth. id="p-28"

[0028] The terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. id="p-29"

[0029] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. Farm animals include, but are not limited to, cattle, goats, pigs, sheep, dogs, horses, and rabbits. Sport animals include, but are not limited to, horses, bovines (calves, bulls, and steers), and dogs. Pet animals include, but are not limited to, dogs, cats, rabbits, rats, pigs, horses, and guinea pigs. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. id="p-30"

[0030] As used herein, the term "administering" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. id="p-31"

[0031] The term "treating" refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment. id="p-32"

[0032] The term "effective amount" or "sufficient amount" refers to the amount of an engineered mammalian cell or other composition that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the physical delivery system in which it is carried. id="p-33"

[0033] For the purposes herein, an effective amount is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect in a subject suffering from cancer or disease. The desired therapeutic effect may include, for example, amelioration of undesired symptoms associated with cancer or disease, prevention of the manifestation of such symptoms before they occur, slowing down the progression of symptoms associated with cancer or disease, slowing down or limiting any irreversible damage caused by cancer or disease, lessening the severity of or curing cancer or disease, or improving the survival rate or providing more rapid recovery from cancer or disease. id="p-34"

[0034] The effective amount depends, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the distribution profile of a therapeutic agent (e.g., a whole-cell cancer vaccine) or composition within the body, the relationship between a variety of pharmacological parameters (e.g., half-life in the body) and undesired side effects, and other factors such as age and gender, etc. id="p-35"

[0035] The term "pharmaceutically acceptable carrier" refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject. "Pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc. In some instances, the carrier is an agent that facilitates the delivery of an engineered mammalian cell to a target cell or tissue. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present disclosure. id="p-36"

[0036] The term "nucleic acid" or "nucleotide" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. id="p-37"

[0037] The term "gene" means the segment of DNA involved in producing a polypeptide chain. The DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons). id="p-38"

[0038] The terms "vector" and "expression vector" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter. The term "promoter" is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators). In the context of the present disclosure, co-expression of multiple genes (e.g., polynucleotides ending an MHC class I allele and/or an MHC class II allele) may be achieved by co-transfection of two or more vectors, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Gene co-expression may be driven by using a plasmid with multiple, individual expression cassettes. Generally, each promoter creates unique mRNA transcripts for each gene that is expressed. Bicistronic or multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. Bicistronic vectors may contain an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. Multicistronic vectors containing one or more self-cleaving 2A peptides are advantageous as they allow gene co-expression from the same cassette. In some instances, multicistronic vectors are preferred when only a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is important. id="p-39"

[0039] "Recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods. A recombinant expression cassette comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide). A recombinant cell is one that has been modified (e.g., transfected or transformed), with a recombinant nucleotide, expression vector or cassette, or the like. id="p-40"

[0040] The term "amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring α-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. "Stereoisomers" of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid). id="p-41"

[0041] Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally-occurring α-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and their combinations. Stereoisomers of a naturally-occurring α-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and their combinations. id="p-42"

[0042] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, "amino acid analogs" can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. "Amino acid mimetics" refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid. Amino acids may be referred to by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. id="p-43"

[0043] The terms "identity," "substantial identity," "similarity," "substantial similarity," "homology" and the related terms and expressions used in the context of describing amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A "comparison window" includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. id="p-44"

[0044] Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993). id="p-45"

[0045] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., alleles), wherein the amino acid residues are linked by covalent peptide bonds. As used herein, the amino acid sequence of a polypeptide is presented from the N-terminus to the C-terminus. In other words, when describing an amino acid sequence of a polypeptide, the first amino acid at the N-terminus is referred to as the "first amino acid." id="p-46"

[0046] The terms "gene editing", "genome editing", "genome engineering", and "genome manipulation" are used interchangeably. These terms refer to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Genome editing can be site-specific. Non-limiting examples of genome editing techniques include the use of nucleases such as clustered regularly interspaced short palindromic repeats/Cas(CRISPR/Cas9) nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). Viral vectors such as integrase-defective lentiviral vectors (IDLVs), adenoviruses and adeno-associated viruses (AAVs) are typically used to deliver DNA for genome editing. Delivery technologies for genome editing are known in the art and any approach may be used for introducing exogenous MHC alleles in the engineered mammalian cells described herein. (See e.g., review by Yin et al., 2017. "Delivery technologies for genome editing." Nature Review Drug Discovery. 16, 387-399). id="p-47"

[0047] The term "cancer" is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, and cancers of all stages and grades including advanced, pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, gynecological cancers (e.g., ovarian, cervical, uterine, vaginal, and vulvar cancers); lung cancers (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, carcinoid tumors, lung adenocarcinoma); breast cancers (e.g., triple-negative breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, Paget’s disease, Phyllodes tumors); digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer (e.g., prostate adenocarcinoma); renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancer (e.g., melanoma); bone and soft tissue sarcomas (e.g., Ewing’s sarcoma); lymphomas; choriocarcinomas; urinary cancers (e.g., urothelial bladder cancer); head and neck cancers; and bone marrow and blood cancers (e.g., acute leukemia, chronic leukemia (e.g., chronic lymphocytic leukemia), lymphoma, multiple myeloma). As used herein, a "tumor" comprises one or more cancerous cells. id="p-48"

[0048] The term of "Major Histocompatibility Complex" or "MHC" refers to a large locus on vertebrate DNA containing a set of closely linked polymorphic genes that code for cell surface proteins essential for the adaptive immune system. id="p-49"

[0049] The MHC locus is present in all jawed vertebrates and contains about a hundred genes and pseudogenes. In humans, the MHC region occurs on chromosome 6, between the flanking genetic markers MOG and COL11A2 (from 6p22.1 to 6p21.3 about 29Mb to 33Mb on the hgassembly) and contains 224 genes spanning 3.6 megabase pairs (3,600,000 bases). In mice, the MHC region occurs on mouse chromosome 17. id="p-50"

[0050] The MHC genes encode MHC molecules/proteins/antigens. The terms "MHC molecules", "MHC proteins", and "MHC antigens" are used interchangeably herein to refer to the cell surface proteins that are encoded by the MHC genes. id="p-51"

[0051] The human MHC is also called the human leukocyte antigen (HLA) complex (often just the HLA). Similarly, the swine MHC is called swine leukocyte antigens (SLA), the bovine MHC is called bovine leukocyte antigens (BoLA), the dog MHC is called dog leukocyte antigens (DLA), etc. The murine MHC is also called the Histocompatibility system 2 or just the H-2. The rat MHC is called RT1, and the chicken MHC is called B-locus. id="p-52"

[0052] The MHC gene family is divided into three groups: MHC class I, MHC class II, and MHC class III. Only MHC class I and class II genes encode the MHC molecules that are directly involved in the antigen presentation. MHC genes are highly polymorphic, for example, HLA class I genes contain 19,031 alleles and HLA class II genes contain 7,183 alleles as listed in the IMGT database. id="p-53"

[0053] The term "allele" refers to a particular form or variant of a gene. Alleles can result from, for example, nucleotide substitutions, additions, or deletions, or can represent a variable number of short nucleotide repeats. In the context of human leukocyte antigen (HLA) genes, HLA alleles are named by the World Health Organization Naming Committee for Factors of the HLA system. Under this system, an HLA gene name is followed by a series of numerical fields. At a minimum, two numerical fields are included. As a non-limiting example, HLA-A*02:101 denotes a specific allele of the HLA-A gene. The first field, separated from the gene name by an asterisk, denotes an allele group. The second field, separated from the first field by a colon, denotes the specific HLA protein that is produced. In some instances, a longer name is used (e.g., HLA-A*02:101:01:02N). In this example, the third numerical field denotes whether a synonymous DNA substitution is present within the coding region, and the fourth numerical field denotes differences between alleles that exist in the non-coding region. In some other instances, an HLA allele name contains a letter at the end. Under the HLA allele naming system, "N" denotes that the allele is a null allele (i.e., the allele produces a non-functional protein), "L" denotes that the allele results in lower than normal cell surface expression of the particular HLA protein, "S" denotes that the allele produces a soluble protein not found on the cell surface, "Q" denotes a questionable allele (i.e., an allele that nay not affect normal expression), "C" denotes that the allele produces a protein that is present in cell cytoplasm but is not present at the cell surface, and "A" denotes an allele that results in aberrant expression (i.e., it is uncertain whether the particular HLA protein is expressed). One of skill in the art will be familiar with the various gene alleles and their naming conventions. id="p-54"

[0054] The term "allele profile" refers to a collection of alleles of one or more genes in a particular sample. The sample may be obtained from a subject, a particular cell or cell type (e.g., a dendritic cell), or from an engineered cell (e.g., a dendritic cell that has been engineered to express one or more proteins). In some instances, an allele profile describes the alleles of a single gene that are present in a sample (e.g., in a cell obtained from a subject or a cell line), or may describe the alleles that are present for two or more genes in a sample. As a non-limiting example, an allele profile may list the alleles that are present for an HLA class II gene in a particular sample. For a diploid cell, only one allele may be present. Alternatively, two different alleles may be present. In other instances, the allele profile enumerates the alleles that are present for two or more genes. id="p-55"

[0055] The term "human leukocyte antigen (HLA)" refers to a gene complex that encodes human major histocompatibility complex (MHC) proteins, which are a set of cell surface proteins that are essential for recognition of foreign molecules by the adaptive immune system. The HLA complex is found within a 3 Mbp stretch of chromosome 6p21. The human MHC Class I proteins, which present peptides from inside the cell, are encoded by the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G genes. HLA-A, HLA-B, and HLA-C genes are more polymorphic, while HLA-E, HLA-F, and HLA-G genes are less polymorphic. HLA-K and HLA-L are also known to exist as pseudogenes. In addition, beta-2-microglobulin is an MHC class I protein, encoded by the (B2M) gene. Non-limiting examples of HLA-A nucleotide sequences are set forth under GenBank reference numbers NM_001242758 and NM_002116. A non-limiting example of an HLA-B nucleotide sequence is set forth under GenBank reference number NM_005514. Non-limiting examples of HLA-C nucleotide sequences are set forth under GenBank reference numbers NM_001243042 and NM_002117. A non-limiting example of an HLA-E nucleotide sequence is set forth under GenBank reference number NM_005516. A non-limiting example of an HLA-F nucleotide sequence is set forth under GenBank reference number NM_018950. A non-limiting example of an HLA-G nucleotide sequence is set forth under GenBank reference number NM_002127. A non-limiting example of a B2M nucleotide sequence is set forth under GenBank reference number NM_004048. id="p-56"

[0056] The human MHC Class II proteins, which present antigens from the outside of the cell to T lymphocytes, are encoded by the HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR genes. HLA-DM genes include HLA-DMA and HLA-DMB. HLA-DO genes include HLA-DOA and HLA-DOB. HLA-DP genes include HLA-DPA1 and HLA-DPB1. HLA-DQ genes include HLA-DQA1, HLA-DQA2, HLA-DQB1, and HLA-DQB2. HLA-DR genes include HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. Non-limiting examples of HLA-DMA and HLA-DMB nucleotide sequences are set forth under GenBank reference numbers NM_006120 and NM_002118, respectively. Non-limiting examples of HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 nucleotide sequences are set forth in GenBank reference numbers NM_01911, NM_002124, NM_022555, NM_021983, NM_002125, respectively. id="p-57"

[0057] The term "semi-allogeneic" refers to at least one MHC class I or class II molecule expressed by a subject’s dendritic cells or a dendritic cell line that is syngeneic to the recipient and at least one MHC class I or class II molecule that is allogeneic to the recipient. "Syngeneic" refers to an MHC allele coding for an MHC molecule specificity that matches between the subject or the dendritic cell line and the recipient and is immunologically compatible with at least one of an MHC class I or class II allele of the recipient. "Allogeneic" refers to at least one of an MHC class I or class II allele coding for an MHC molecule specificity that is unmatched and immunologically incompatible with at least one of an MHC class I or class II allele of the recipient. id="p-58"

[0058] The term "vaccine" refers to a biological composition that, when administered to a subject, has the ability to produce an acquired immunity to a particular pathogen or disease in the subject. Typically, one or more antigens, or fragments of antigens, that are associated with the pathogen or disease of interest are administered to the subject. Vaccines can comprise, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins that are expressed from or on cells (e.g., cell surface proteins), proteins that are produced by organisms (e.g., toxins), or portions of organisms (e.g., viral envelope proteins). In some instances, cells are engineered to express proteins such that, when administered as a vaccine, they enhance the ability of a subject to acquire immunity to that particular cell type (e.g., enhance the ability of a subject to acquire immunity to a cancer cell). As used herein, the term "vaccine" or "whole-cell cancer vaccine" includes but is not limited to the engineered mammalian cell(s) of the present disclosure. id="p-59"

[0059] The term "cytokine" refers to small proteins released by cells that have a specific effect on the interactions and communications between cells. Cytokines are generally known as lymphokines (e.g., cytokines made by lymphocytes), monokines (e.g., cytokines made by monocytes), or chemokines (e.g., cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (e.g., autocrine action), on nearby cells (e.g., paracrine action), or on distant cells (e.g., endocrine action). In the context of the present disclosure, cytokines may comprise a chemokine, an interferon, an interleukin, and/or a tumor necrosis factor (TNF). As an example, cytokines may comprise an early T cell activation antigen-1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36Ƴ, IL-37, IL-38), an interleukin-(IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-(IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-(IL-10), an interleukin-12 (IL-12), an interleukin-13 (IL-13), an interleukin-15 (IL-15), an interleukin-17 (IL-17), an interleukin-18 (IL-18), an interleukin-23 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), a type I interferon family member (IFN-α, IFNβ, IFNε, IFNκ, IFNω), a type II interferon family member (IFNγ), a type III interferon family member (IFNλ1 (IL-29), IFNλ2 (IL-28A), IFNλ3 (IL-28B), IFNλ4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-1α, MIP-1β), a lymphotactin, and/or a fractalkine. id="p-60"

[0060] The term "granulocyte macrophage colony-stimulating factor (GM-CSF)" refers to a monomeric glycoprotein also known as "colony stimulating factor (CSF2)" that is secreted by cells such as macrophages, T cells, mast cells, natural killer (NK) cells, endothelial cells, and fibroblasts. GM-CSF functions as a cytokine that affects a number of cell types, in particular macrophages and eosinophils. As part of the immune/inflammatory cascade, GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) and monocytes. The monocytes subsequently mature into macrophages and dendritic cells after tissue infiltration. A non-limiting example of a CSF2 nucleotide sequence (the gene that encodes GM-CSF) in humans is set forth under GenBank reference number NM_000758. id="p-61"

[0061] The term "interferon" refers to a cytokine that is produced in response to infection or other inflammatory stimuli. Interferons are signaling proteins that are synthesized and released by host cells in response to a pathogen (e.g., viruses, bacteria, parasites, tumor cells). Interferons are classified into three subgroups: type I interferons, type II interferon (IFNγ), and type III interferons. Functionally, these cytokines modulate immune cell function. Although type III interferons are structurally distinct from type I interferons, they have overlapping functions, and both signal through the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway to induce transcription of interferon-stimulated genes (ISGs) and promote immune responses. (See e.g., Goel et al. (2021). Interferon lambda in inflammation and autoimmune rheumatic diseases. Nat Rev Rheumatol 17, 349–362). Type I interferon proteins include IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, IFN-δ, IFN-ζ, IFN-ω, and IFN-ν. Interferon alpha proteins are produced by leukocytes and are mainly involved in the innate immune response. Genes that encode IFN-α proteins include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. Non-limiting examples of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21 human nucleotide sequences are set forth in Gene Bank reference numbers NM_024013, NM_000605, NM_021068, NM_002169, NM_021002, NM_021057, NM_002170, NM_002171, NM_006900, NM_002172, NM_002173, NM_021268, and NM_002175, respectively. As an example, the gene IFNA2 encodes IFN-α2a, IFN-α2b, and IFN-α2c variants. As used herein, the terms "IFN-α" and "IFN-α2" are used interchangeably, and they refer to interferon proteins IFN-α2a or IFN-α2b. Type III interferon proteins include interferon lambda 1 (IFNλ1 (IL-29), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 1 (IFNλ3 (IL-28B)), and interferon lambda 4 (IFNλ4). Interferon lambda family members signal through the common IL-10 receptor subunit 2 (IL-10R2). Human interferon lambda proteins are encoded by four IFNL genes, IFNL(IL29), IFNL2 (IL28A), IFNL3 (IL28B), and IFNL4. id="p-62"

[0062] The term "co-stimulatory molecule" refers to a cell surface molecule that amplifies or counteracts the initial activating signals provided to T cells from the T cell receptor (TCR) following its interaction with an antigen/major histocompatibility complex (MHC). Co-stimulatory molecules generally may influence T cell differentiation and fate. Co-stimulatory molecules belong to three major families, namely the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF) – TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family. (See e.g., Rodriguez-Manzanet, Roselynn et al. "The costimulatory role of TIM molecules." Immunological reviews vol. 229,1 (2009): 259-70.) Exemplary co-stimulatory molecules and ligands include, but are not limited to, CD28 and ligands B7-1 (CD80), CTLA-4, PDL-1, or B7-2 (CD86), CTLA-4 and ligands B7-1 (CD80) or B7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (a.k.a. CD154), OX40 and ligand OX40L, GITR and ligand GITRL, TIM-1 and ligands TIM-1, TIM-4, IgA, or phosphatidylserine (PtdSer), TIM-2 and ligands H-ferritin or semaphorin 4A (Sem4A), and TIM-4 and ligand phosphatidylserine (PtdSer). In the context of the present disclosure, co-stimulatory molecules may comprise a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), OX40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), CD30 molecule (CD30), and combinations thereof (See e.g., FIG. 5 ). id="p-63"

[0063] " Regulatory T cells" or "Tregs" or "suppressor T cells" refer to a highly immunosuppressive subset of CD4+CD25+ T cells that are essential to establish and maintain homeostasis and self-tolerance. Tregs can inhibit T cell proliferation and cytokine production and prevent autoimmunity. Treg cells express the biomarkers CD4, CD25, and Forkhead box protein P3 (FoxP3). CD4 is a marker for some thymic-derived populations of Tregs in addition to helper T cells. CD25 is a component of the IL-2 receptor and can serve as a marker for activated T cells. FoxP3 is a transcription factor that represses the expression of several cytokines such as IL-2, IL- 4 and IFNγ with concomitant activation of the IL-2 receptor (CD25), cytotoxic T lymphocyte–associated protein-4 (CTLA4) and glucocorticoid-induced TNF receptor (GITR). FoxP3 is the master protein involved in the differentiation and functional expression of regulatory T cells and can be used as a Treg molecular marker (see, e.g, Science (2003) 299: 1057-61). Further description and information on human CD4+CD25+ regulatory T cells can be found in the following references, which are all hereby incorporated by reference: Jonuleit et al. (2001) J Exp Med. 193:1285-94; Levings et al. (2001) J Exp Med 193:1295-1301; Dieckmann et al. (2001) J Exp Med 193:1303-1310; and Yamagiwa et al. (2001) J. Immunol. 166:7282-89, Stephens et al. (2001) Eur. J. Immunol. 31:1247-1254; and Taams et al. (2001) Eur. J. Immunol. 31:1122-1131. id="p-64"

[0064] The terms "regulatory T cell inhibitory agent", "Treg inhibitory agent", and "Treg agent" refer to an agent that: (1) inhibits or decreases the activity or function of a regulatory T cell; (2) decreases the population of regulatory T cells in a subject (in one embodiment, the decrease can be temporary, for example, for a few hours, a day, a few days, a week, or a few weeks); or (3) substantially ablates or eliminates the population of regulatory T cells in a subject (in one embodiment, the ablation or elimination can be temporary, for example, for a few hours, a day, a few days, a week, or a few weeks). A Treg agent can decrease the suppression of immune system activation and can decrease prevention of self-reactivity. Exemplary Treg agents include, but are not limited to, a compound, antibody, fragment of an antibody, or chemical that targets a Treg cell surface marker (such as CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), CTLA4, CCR4, CCR8, FOXP3, LAG3, CD103, NRP-1, glucocorticoid-induced TNF receptor (GITR), galectin-1, TNFR2, or TGF-βR1). In certain embodiments, a Treg agent targets a Treg cell surface marker that is involved in Treg activation such that the Treg inhibitor prevents Treg activation. Exemplary Treg agents include, but are not limited to, antibodies, fusion proteins, ONTAK, HuMax-Tac, Zenapax, or MDX-010, aptamers, siRNA, ribozymes, antisense oligonucleotides, and the like. The administration of a Treg agent or derivatives thereof can block the action of its target, such as a Treg cell surface marker. A Treg agent can have an attached toxic moiety such that upon internalization of the inhibitor, the attached toxic moiety can kill the regulatory T cell. id="p-65"

[0065] The term "tumor antigen" refers to an antigenic substance produced in tumor cells that may trigger an immune response in the host. Tumor antigens generally refer to tumor-associated antigen (TAAs) or tumor-specific antigens (TSAs). Typically, TSAs are found in cancer cells only and are not in healthy (e.g., non-cancerous) cells. TSAs may arise from oncogenic driver mutations that generate novel peptide sequences (e.g., neoantigens). A non-limiting example of a TSA is alphafetoprotein (AFP) expressed in germ cell tumors and hepatocellular carcinoma. TAAs have elevated levels in tumor cells and may express at lower levels in healthy cells. A non-limiting example of a TAA is melanoma-associated antigen (MAGE) expressed in the testis along with malignant melanoma. id="p-66"

[0066] The term "survival" refers to a length of time following the diagnosis of a disease and/or beginning or completing a particular course of therapy for a disease (e.g., cancer). The term "overall survival" includes the clinical endpoint describing patients who are alive for a defined period of time after being diagnosed with or treated for a disease, such as cancer. The term "disease-free survival" includes the length of time after treatment for a specific disease (e.g., cancer) during which a patient survives with no sign of the disease (e.g., without known recurrence). In certain embodiments, disease-free survival is a clinical parameter used to evaluate the efficacy of a particular therapy, which is usually measured in units of 1 or 5 years. The term "progression-free survival" includes the length of time during and after treatment for a specific disease (e.g., cancer) in which a patient is living with the disease without additional symptoms of the disease. In some embodiments, survival is expressed as a median or mean value.

III. Detailed Description of the Embodiments id="p-67"

[0067] In one aspect, the present disclosure provides an engineered mammalian dendritic cell comprising one or more exogenous alleles of an MHC class II gene. In some embodiments, the engineered mammalian dendritic cell comprises one exogenous allele of an MHC class II gene. In other embodiments, the engineered mammalian dendritic cell comprises more than one exogenous MHC class II allele. In some instances, the engineered mammalian dendritic cell comprises a first exogenous allele of a first MHC class II gene and a second exogenous allele of a second MHC class II gene. In some instances, the engineered mammalian dendritic cell comprises a first exogenous allele of an MHC class II gene and a second exogenous allele of the same MHC class II gene. In some embodiments, the engineered mammalian dendritic cell further comprises one or more exogenous alleles of an MHC class I gene. id="p-68"

[0068] In one aspect, the one or more exogenous alleles are introduced into the mammalian dendritic cell through homologous recombination. In some embodiments, one or more exogenous MHC class II alleles are introduced into the mammalian dendritic cell through homologous recombination. In some instances, the homologous recombination replaces an endogenous allele of the MHC class II gene with an exogenous allele. In other instances, the homologous recombination inserts an exogenous allele into the MHC class II gene without deleting the related endogenous allele. In other embodiments, one or more exogenous MHC class I alleles are also introduced through homologous recombination into the mammalian dendritic cell. id="p-69"

[0069] In some embodiments, the homologous recombination is triggered by a nuclease creating a double-stranded break (DSB) at a specific site in the genome. The nuclease can be an endonuclease, a Zinc finger nuclease (ZEN), a transcription activator-like effector nuclease (TALEN), site-specific recombinase, transposase, topoisomerase, and modified derivatives and variants thereof. Descriptions of nucleases that can be used in the present disclosure are provided further herein. In some embodiments, the nuclease can be an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease. id="p-70"

[0070] In another aspect, the one or more exogenous alleles are introduced through transfection or transduction of one or more expression vectors into the cell. In some embodiments, one or more exogenous MHC class II alleles are introduced through transfection or transduction of one or more expression vectors into the cell. In some instances, one or more exogenous MHC class II alleles are introduced through transfection of one or more expression vectors into the cell. In some embodiments, transfection of one or more expression vectors comprising MHC class II alleles into the cell includes transfection of plasmids or expression cassettes. In other instances, one or more exogenous MHC class II alleles are introduced through transduction of one or more expression vectors into the cell. In some embodiments, transduction of one or more expression vectors comprising MHC class II alleles into the cell includes transduction of viral vectors such as, but not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors. In some embodiments, only one exogenous MHC class II allele is introduced through transfection or transduction of one vector into the cell. In other embodiments, more than one exogenous MHC class II alleles are introduced into the cell. In some instances, all the exogenous MHC class II alleles can be present on the same vector. In other instances, each exogenous MHC class II allele can be present on a separate vector. In yet other instances, two, three, four, five, six, or more exogenous MHC class II alleles can be present on the same vector. Any number of combinations of exogenous alleles on a single vector and any number of vectors in a cell is permitted. Descriptions of expression vectors and the methods of transfection or transduction that can be used in the present disclosure are provided further herein. In certain embodiments, two exogenous MHC class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles are introduced into the cell on the same vector or separate vectors. In other embodiments, one or more exogenous MHC class I alleles are also introduced through transfection or transduction of one or more expression vectors into the mammalian dendritic cell. In certain instances, all the exogenous MHC class II alleles can be present on one vector, and all the exogenous MHC class I alleles can be present on another vector. id="p-71"

[0071] In one aspect, the engineered mammalian dendritic cell described herein is an engineered human dendritic cell. In other aspects, the engineered mammalian dendritic cell is derived from a non-human dendritic cell. In some embodiments, the non-human dendritic cell can be from a mouse, a rat, a simian, a farm animal, a sport animal, or a pet animal. Non-limiting examples of farm animals include cattle, goats, pigs, sheep, dogs, horses, and rabbits. Non-limiting examples of sport animals include horses, bovines (calves, bulls, and steers), and dogs. Non-limiting examples of pet animals include dogs, cats, rabbits, rats, pigs, horses, and guinea pigs.

A. Human MHC genes HLA class II genes [0072] In one aspect, the present disclosure provides an engineered human dendritic cell comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous HLA class II alleles of an MHC class II gene or a plurality thereof. In some embodiments, the MHC class II gene is an HLA class II alpha subunit gene. In other embodiments, the MHC class II gene is an HLA class II beta subunit gene. In certain embodiments, the MHC class II gene is a combination of HLA class II alpha subunit and HLA class II beta subunit genes. id="p-73"

[0073] In other embodiments, the MHC class II gene is an HLA-DR gene, an HLA-DP gene, an HLA-DQ gene, an HLA-DM gene, and/or an HLA-DO gene. id="p-74"

[0074] In some embodiments, the HLA-DR gene is an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, and/or an HLA-DRB5 gene. In certain embodiments, the engineered human dendritic cell comprises one or more exogenous alleles of one or more (e.g., 1, 2, 3, 4, 5, or more) HLA-DR gene(s). In some instances, the HLA-DP gene is an HLA-DPAgene. In other instances, the HLA-DP gene is an HLA-DPB1 gene. In certain instances, the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DPA1 and HLA-DPB1 gene alleles. In some instances, the HLA-DQ gene is an HLA-DQA1 gene. In other instances, the HLA-DQ gene is an HLA-DQB1 gene. In certain instances, the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DQA1 and HLA-DQB1 gene alleles. In some instances, the HLA-DM gene is an HLA-DMA gene. In other instances, the HLA-DM gene is an HLA-DMB gene. In certain instances, the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DMA and HLA-DMB gene alleles. In some instances, the HLA-DO gene is an HLA-DOA1 gene. In other instances, the HLA-DO gene is an HLA-DOB1 gene. In certain instances, the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DOA1 and HLA-DOB1 gene alleles. id="p-75"

[0075] Examples of suitable HLA-DRB3 alleles include but are not limited to HLA-DRB3*02:02, HLA-DRB3*01:01, and HLA-DRB3*03:01. Examples of suitable HLA-DRBalleles include but are not limited to HLA-DRB4*01:01 and HLA-DRB4*01:03. Examples of suitable HLA-DRB5 alleles include but are not limited to HLA-DRB5*01:02, HLA-DRB5*01:01, and HLA-DRB5*02:02. The engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, or more) exogenous alleles of HLA-DRB3/4/5 alleles. id="p-76"

[0076] Examples of suitable HLA-DPA1 alleles include but are not limited to HLA-DPA1*01:05, HLA-DPA1 *02:08, and HLA-DPA1 *04:05. Examples of suitable HLA-DPBalleles include but are not limited to HLA-DPB1 *32:01 and HLA-DPB1 *1454:01. Examples of suitable HLA-DQA1 alleles include but are not limited to HLA-DQA1*01:06, HLA-DQA1*02:29, and HLA-DQA1*06:04. Examples of suitable HLA-DQB1 alleles include but are not limited to HLA-DQB1*05:04, HLA-DQB1*06:100, and HLA-DQB1*04:95. Examples of suitable HLA-DMA alleles include but are not limited to HLA-DMA*01:01:01:01, HLA-DMA*01:01:02, and HLA-DMA*01:02:01:09. Examples of suitable HLA-DQB alleles include but are not limited to HLA-DMB*01:03:01:05, HLA-DMB*01:01:01:20, and HLA-DMB*01:01:01:01.

HLA class I genes [0077] In one aspect, the present disclosure provides an engineered human dendritic cell further comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous HLA class I alleles of an MHC class I gene or a plurality thereof. In some embodiments, the MHC class I gene is an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, or a B2M gene. In other embodiments, the MHC class I gene is a combination of an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, and/or a B2M gene. id="p-78"

[0078] Examples of suitable HLA-A alleles include but are not limited to HLA-A*11:01, HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*26:01, HLA-A*29:02, HLA-A*32:01, HLA-A*24:02, HLA-A*33:03, HLA-A*68:01, HLA-A*31:01, and HLA-A*02:06. Engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) exogenous HLA-A alleles. id="p-79"

[0079] Examples of suitable HLA-B alleles include but are not limited to HLA-B*13:02, HLA-B*41:01, HLA-B*18:03, HLA-B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51:01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01, and HLA-B*52:01. Engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more) exogenous HLA-B alleles. id="p-80"

[0080] Examples of suitable HLA-C alleles include but are not limited to HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01:02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C*12:03, and HLA-C*14:02. Engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) exogenous HLA-C alleles.

B. Murine MHC genes id="p-81"

[0081] Murine MHC is composed of 11 subclasses. The "classical MHC class I" (also called MHC-Ia comprises H-2D, H-2K and H-2L subclasses located in the K region and D region of the murine H-2 loci ( FIG. 2D ). The "non-classical MHC class I" (MHC- Ib) comprises H-2Q, H-2M and H-2T subclasses in the Q/T/M region. The "classical MHC class II" (MHC-IIa) includes H-2A(I-A), and H-2E(I-E) subclasses, and the "non-classical MHC class II" (MHC-IIb) comprises H-2P (P), H-2M (DM) and H-2O (DO). All murine MHC class II subclasses are located in the I region. The murine MHC class III is located in the S region. id="p-82"

[0082] Murine MHC class I molecules consist of a 45 kD highly glycosylated heavy chain non-covalently associated with a 12 kD 2-microglobulin, a polypeptide that is also found free in serum. Murine MHC class II antigen is composed of a 33 kD chain and a 28 kD chain. id="p-83"

[0083] MHC class I molecules are expressed on almost all nucleated cells. They play an important role in presentation of altered self-cell antigens (virally infected or tumor cells) to CD8+ cytotoxicity T cells. The MHC class II molecules are expressed on antigen presenting cells (B cells, monocytes/ macrophages, dendritic cells, and Langerhans cells, etc.). They are involved in presentation of processed peptide antigens to CD4+ cells. id="p-84"

[0084] In one aspect, the present disclosure provides an engineered murine dendritic cell comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous MHC class II alleles of an MHC class II gene or a plurality thereof. In another aspect, the engineered murine dendritic cell further comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous MHC class I alleles of an MHC class I gene or a plurality thereof.

MHC haplotypes in murine strains [0085] Laboratory mice are inbred so that each strain is homozygous and has a unique MHC haplotype. The MHC haplotype in these strains is designated by a small letter (a, b, d, k, q, s, etc.). For example, MHC haplotype antigens of BALB/c mice are H-2Kd, H-2Dd, H-2Ld, H2-IAd, and H2-IEd and MHC haplotype antigens of C57BL/6J mice are H2-Db, H2-Kb, and H2-IAb. Some variant murine strains are also generated in the laboratory. For example, B6.C-H2-Kbm1/ByJ (bm1) is generated through the introgression of a spontaneous variant (BALB/cBy x C57BL/6By)F1-derived H2bm1 allele on chromosome 17 onto the C57BL/6By background for 10 generations. The variant allele, H2-Kbm1, differs from H2-Kb by 7 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 152, 155, and 156 of the αdomain. B6(C)-H2-Ab1bm12/KhEgJ (bm12) is also generated through the introgression of a spontaneous variant (C57BL/6Kh x BALB/cKh) F1-derived H2-Ab1bm12 allele onto the C57BL/6Kh background for 10 generations. This variant MHC class II allele differs from H2-Ab1b by 3 nucleotides resulting in 3 amino acid substitutions (Ile67Phe, Arg70Gln, Thr71Lys) occurring along the edge of the peptide binding groove of the β1 domain.

C. Selected MHC allele constructs id="p-86"

[0086] In some embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) comprises one or more exogenous MHC class II genes selected from any MHC class II gene, a codon optimized version thereof, a variant thereof, or a fragment thereof. For instance, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more MHC class II genes driven by one or more promoters. In some embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises one or more exogenous MHC class I genes selected from any class I gene, a codon optimized version thereof, a variant thereof, or a fragment thereof. For instance, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more MHC class I genes driven by one or more promoters. [0087] In some embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) comprises one exogenous MHC class II gene selected from any MHC class II gene, a codon optimized version thereof, a variant thereof, or a fragment thereof. In other embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) comprises at least two exogenous MHC class II gene selected from any class II gene, a codon optimized version thereof, a variant thereof, or a fragment thereof. For instance, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) may comprise a first recombinant polynucleotide encoding a first MHC class II gene and a second recombinant polynucleotide encoding a second MHC class II gene. id="p-88"

[0088] In some embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises one exogenous MHC class I gene selected from any MHC class I gene, a codon optimized version, a variant thereof, or a fragment thereof. In other embodiments, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises at least two recombinant polynucleotides each encoding an MHC class I gene selected from any MHC class I gene, a codon optimized version, a variant thereof, or a fragment thereof. For instance, the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) may comprise a recombinant polynucleotide encoding a first MHC class I gene and a second recombinant polynucleotide encoding a second MHC class I gene.

D. Cytokines and/or co-stimulatory molecules id="p-89"

[0089] In one aspect, the present disclosure provides an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprising one or more recombinant polynucleotides encoding a cytokine, a co-stimulatory molecule, a variant thereof, a fragment thereof, or a combination thereof. id="p-90"

[0090] The cytokine can be a chemokine, an interferon, an interleukin, or a tumor necrosis factor. The cytokine can be selected from an early T cell activation antigen-1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36Ƴ, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-(IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-(IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin-10 (IL-10), an interleukin-(IL-12), an interleukin-13 (IL-13), an interleukin-15 (IL-15), an interleukin-17 (IL-17), an interleukin-18 (IL-18), an interleukin-21 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), an interferon alpha (IFN-α), an interferon lambda 1 (IFNλ1 (IL-29)), an interferon lambda 2 (IFNλ2 (IL-28A)), an interferon lambda 3 (IFNλ3 (IL-28B)), an interferon lambda 4 (IFNλ4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-1α, MIP-1β), a lymphotactin, or a fractalkine. id="p-91"

[0091] The co-stimulatory molecule can be selected from at least one of a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL, also known as TNFSF9 or CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CDmolecule (CD40), OX40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), and a combination thereof. id="p-92"

[0092] In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be introduced into the cell through homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be introduced into the cell through transfection or transduction of one or more expression vectors. id="p-93"

[0093] In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules are present on one or more vectors in the cell and one or more exogenous MHC alleles are in the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules are in the genome of the cell and one or more exogenous MHC alleles are present on one or more vectors in the same cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules and one or more exogenous MHC alleles are in the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules and one or more exogenous MHC alleles are present on one or more vectors in the cell. id="p-94"

[0094] In some instances, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be present on the same vector with one or more exogenous MHC alleles. In other instances, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be present on a separate vector with one or more exogenous MHC alleles. As a non-limiting example, the cell comprises: (a) a vector comprising one or more exogenous HLA class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles; and (b) a vector comprising one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules. As another non-limiting example, the cell further comprises: (c) a vector comprising one or more exogenous HLA class I alleles.

E. Heterologous antigens id="p-95"

[0095] In one aspect, the present disclosure provides the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprising one or more recombinant polynucleotides encoding a heterologous antigen (e.g., an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an immune response), a variant thereof, or a fragment thereof. id="p-96"

[0096] In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be introduced into the cell through homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be introduced into the cell through transfection or transduction of one or more expression vectors. id="p-97"

[0097] In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide are present on one or more vectors in the cell and one or more exogenous MHC alleles are in the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide are in the genome of the cell and one or more exogenous MHC alleles are present on one or more vectors in the same cell. In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide and one or more exogenous MHC alleles are in the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide and one or more exogenous MHC alleles are present on one or more vectors in the cell. id="p-98"

[0098] In some instances, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be present on the same vector with the exogenous MHC alleles. In other instances, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be present on a separate vector with the exogenous MHC alleles. As a non-limiting example, the cell comprises: (a) a vector comprising one or more HLA class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles; and (b) a vector comprising recombinant polynucleotides encoding a unique antigen peptide of a pathogenic antigen, a tumor-associated antigen, a neo-antigen, an allergen, or an antigen that is the target of an immune response. As another non-limiting example, the cell further comprises: (c) a vector comprising one or more exogenous HLA class I alleles (e.g., HLA-A alleles). id="p-99"

[0099] In another aspect, the present disclosure provides an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprising one or more heterologous antigens (e.g., an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an immune response), a variant thereof, or a fragment thereof. In some embodiments, one or more antigens and/or an antigen peptide can be introduced into the cell through incubation of the antigen and/or the antigen peptide with the cell in the same culture. id="p-100"

[0100] In some embodiments, an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) is derived from a primary patient cell and pulsed by a heterologous antigen and/or an antigen peptide of a pathogen that the patient is suffering from. In some embodiments, an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) is derived from a cancer patient and pulsed by a tumor-specific antigen and/or a fragment thereof. In some embodiments, an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) derived from a patient primary cell is further incubated with Lipopolysaccharide (LPS) after pulsing.

F. Dendritic cells id="p-101"

[0101] In some embodiments, the engineered mammalian dendritic cell is derived from a dendritic cell line (e.g., a human dendritic cell line). Non-limiting examples of human dendritic cell lines include the following cell lines and subclones thereof: HL-60, THP-1, K562, MUTZ3, or an immortalized dendritic cell. In some instances, the immortalized dendritic cell expresses HTLV-1 transactivator (Tax) protein, SV40 proteins, and/or hTERT. id="p-102"

[0102] In other embodiments, the engineered mammalian dendritic cell is engineered from a primary cell of a patient, for example, a blood or biopsy sample from a patient. In some instances, the patient has a cancer.

G. Homologous recombination id="p-103"

[0103] In one aspect, the engineered mammalian dendritic cell described herein contains one or more exogenous alleles in the genomic DNA. In some embodiments, the one or more exogenous MHC class II alleles are introduced into the cell through homologous recombination. In some embodiments, the homologous recombination replaces an endogenous allele of the MHC class II gene with an exogenous allele. In other embodiments, the homologous recombination inserts an exogenous allele into the MHC class II gene. id="p-104"

[0104] As described herein, a nuclease can be used to create a double-stranded break (DSB) at a specific site in the genome and trigger homologous recombination. Examples of nucleases include, but are not limited to, an endonuclease, a Zinc finger nuclease (ZEN), a transcription activator-like effector nuclease (TALEN), site-specific recombinase, transposase, topoisomerase, and modified derivatives and variants thereof. In some embodiments, the nuclease can be an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease.

Cas Endonuclease [0105] In some embodiments, the nuclease used in methods and compositions of the disclosure is a CRISPR-associated (Cas) protein. A Cas protein refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. A Cas protein can induce double strand breaks in genomic DNA (target nucleic acid) when both functional domains are active. The Cas protein can comprise one or more catalytic domains of a Cas protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the Cas protein can be a fusion protein, e.g., the two catalytic domains are derived from different bacteria species. id="p-106"

[0106] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, homologs thereof, variants thereof, mutants thereof, and derivatives thereof. There are three main types of Cas proteins (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(1):58-66). Type II Cas proteins include Cas1, Cas2, Csn2, Cas9, and Cfp1. These Cas proteins are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP_269215, and the amino acid sequence of Streptococcus thermophilus wild-type Caspolypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470. id="p-107"

[0107] Cas proteins, e.g., Cas9 nucleases, can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida. id="p-108"

[0108] In some embodiments, the Cas protein can be a high-fidelity or enhanced specificity Caspolypeptide variant with reduced off-target effects and robust on-target cleavage. Non-limiting examples of Cas9 polypeptide variants with improved on-target specificity include the SpCas(K855A), SpCas9 (K810A/K1003A/R1060A) (also referred to as eSpCas9(1.0)), and SpCas(K848A/K1003A/R1060A) (also referred to as eSpCas9(1.1)) variants described in Slaymaker et al., Science, 351(6268):84-8 (2016), and the SpCas9 variants described in Kleinstiver et al., Nature, 529(7587):490-5 (2016) containing one, two, three, or four of the following mutations: N497A, R661A, Q695A, and Q926A (e.g., SpCas9-HF1 contains all four mutations).

Guide RNAs [0109] A Cas protein can be guided to its target nucleic acid by a guide RNA (gRNA). A gRNA is a version of the naturally occurring two-piece guide RNA (crRNA and tracrRNA) engineered into a two-piece gRNA or a single, continuous sequence. A gRNA can contain a guide sequence (e.g., the crRNA equivalent portion of the gRNA) that targets the Cas protein to the target nucleic acid and a scaffold sequence that interacts with the Cas protein (e.g., the tracrRNAs equivalent portion of the gRNA). A gRNA can be selected using a software. As a non-limiting example, considerations for selecting a gRNA can include, e.g., the PAM sequence for the Cas protein to be used, and strategies for minimizing off-target modifications. Tools, such as NUPACK® and the CRISPR Design Tool, can provide sequences for preparing the gRNA, for assessing target modification efficiency, and/or assessing cleavage at off-target sites.

Guide Sequence [0110] The guide sequence in the gRNA may be complementary to a specific sequence within a target nucleic acid (e.g., one allele of an MHC Class II gene). The 3’ end of the target nucleic acid sequence can be followed by a PAM sequence. Approximately 20 nucleotides upstream of the PAM sequence is the target nucleic acid. In general, a Cas9 protein or a variant thereof cleaves about three nucleotides upstream of the PAM sequence. The guide sequence in the gRNA can be complementary to either strand of the target nucleic acid. id="p-111"

[0111] In some embodiments, the guide sequence of a gRNA comprises about 100 nucleic acids at the 5’ end of the gRNA that can direct the Cas protein to the target nucleic acid site using RNA-DNA complementarity base pairing. In some embodiments, the guide sequence comprises about nucleic acids at the 5’ end of the gRNA that can direct the Cas protein to the target nucleic acid site using RNA-DNA complementarity base pairing. In other embodiments, the guide sequence comprises less than 20, e.g., 19, 18, 17, 16, 15 or less, nucleic acids that are complementary to the target nucleic acid site. In some instances, the guide sequence in the gRNA contains at least one nucleic acid mismatch in the complementarity region of the target nucleic acid site. In some instances, the guide sequence contains about 1 to about 10 nucleic acid mismatches in the complementarity region of the target nucleic acid site.

Scaffold Sequence [0112] The scaffold sequence in the gRNA can serve as a protein-binding sequence that interacts with the Cas protein or a variant thereof. In some embodiments, the scaffold sequence in the gRNA can comprise two complementary stretches of nucleotides that hybridize to one another to form a double-stranded RNA duplex (dsRNA duplex). The scaffold sequence may have structures such as lower stem, bulge, upper stem, nexus, and/or hairpin. In some embodiments, the scaffold sequence in the gRNA can be between about 90 nucleic acids to about 120 nucleic acids.

Zinc Finger Nuclease [0113] In some embodiments, the nuclease is a Zinc-finger nuclease (ZFN). ZFNs typically comprise a zinc finger DNA binding domain and a nuclease domain. Generally, ZFNs include two zinc finger arrays (ZFAs), each of which is fused to a single subunit of a non-specific endonuclease, such as the nuclease domain from the FokI enzyme, which becomes active upon dimerization. Typically, a single ZFA consists of 3 or 4 zinc finger domains, each of which is designed to recognize a specific nucleotide triplet (GGC, GAT, etc.). A ZFN composed of two "3-finger" ZFAs is therefore capable of recognizing an 18 base pair target site (i.e., recognition sequence); an 18 base pair recognition sequence is generally unique, even within large genomes such as those of humans and plants. By directing the co-localization and dimerization of the two FokI nuclease monomers, ZFNs generate a functional site-specific endonuclease that can target a particular locus (e.g., gene, promotor or enhancer). id="p-114"

[0114] Zinc-finger nucleases useful in the methods disclosed herein include those that are known and ZFN that are engineered to have specificity for one or more target sites described herein (e.g., promotor or enhancer nucleotide sequence). Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence within a target site of the host cell genome. ZFN can comprise an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example, a nuclease domain from a Type IIs endonuclease such as HO or FokI. In some examples, a zinc finger DNA binding domain can be fused to a site-specific recombinase, transposase, or a derivative thereof that retains DNA nicking and/or cleaving activity. id="p-115"

[0115] In some embodiments, additional functionalities can be fused to the zinc-finger binding domain, including but not limited to, transcriptional activator domains (such as VP16, VP48, VP64, VP160 and the like) or transcription repressor domains (such as KRAB). In one embodiment, the zinc finger nuclease is engineered such that the zinc finger nuclease comprises a transcriptional activator domain selected from VP16, VP48, VP64 or VP160. In one embodiment, the zinc finger nuclease is engineered such that the zinc finger nuclease comprises a transcriptional activator domain selected from HSF1, VP16, VP64, p65, RTA, MyoD1, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9. For example, engineered zinc finger transcriptional activator that interact with a promoter region of the gamma-globulin gene was shown to enhance fetal hemoglobin production in primer adult erythroblasts (Wilber et al., Blood, 115(15):3033-3041). Other polydactyl zinc-finger transcription factors are also known in the art, including those disclosed in Beerli and Barbas (see, Nature Technology, (2002) 20:135-141). id="p-116"

[0116] Each zinc finger domain recognizes three consecutive base pairs in the target DNA. For example, a three finger domain recognizes a sequence of nine contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind a nucleotide recognition sequence. Useful zinc finger modules include those that recognize various GNN and ANN triplets (Dreier et al., (2001) J Biol Chem 276:29466-78; Dreier et al., (2000) J Mol Biol 303:489-502; Liu et al., (2002) J Biol Chem 277:3850-6), as well as those that recognize various CNN or TNN triplets (Dreier et al., (2005) J Biol Chem 280:35588-97; Jamieson et al., (2003) Nature Rev Drug Discovery 2:361-8). See also, Durai et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002) Methods 26:76-83; Porteus and Carroll, (2005) Nat Biotechnology 23:967-73; Pabo et al., (2001) Ann Rev Biochem 70:313-40; Wolfe et al., (2000) Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas (2001) Curr Opin Biotechnol 12:632-7; Segal et al., (2003) Biochemistry 42:2137-48; Beerli and Barbas, (2002) Nat Biotechnol 20:135-41; Carroll et al., (2006) Nature Protocols 1:1329; Ordiz et al., (2002) Proc Natl Acad Sci USA 99:13290-5; Guan et al., (2002) Proc Natl Acad Sci USA 99:13296-301; WO2002099084; WO00/42219; WO02/42459; WO2003062455; US20030059767; US Patent Application Publication Number 2003/0108880; U.S. Pat. Nos. 6,140,466, 6,511,808 and 6,453,242. Useful zinc-finger nucleases also include those described in WO03/080809; WO05/014791; WO05/084190; WO08/021207; WO09/042186; WO09/054985; and WO10/065123. id="p-117"

[0117] In some embodiments, a ZFN comprises a fusion protein having a cleavage domain of a Type IIS restriction endonuclease fused to an engineered zinc finger binding domain, wherein the binding domain further comprises one or more transcriptional activators. In some embodiments, the type IIS restriction endonuclease is selected from a HO endonuclease or a FokI endonuclease. In some embodiments, the zinc finger binding domain comprises 3, 4, 5 or 6 zinc fingers. In another embodiment, the zinc finger binding domain specifically binds to a recognition sequence corresponding to a promoter or enhancer disclosed herein (e.g., SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A and PAX6 promotor or enhancer). In one embodiment, the one or more transcriptional activators is selected from VP16, VP48, VP64, or VP160. Generally, the DNA-binding domain of a ZFN contains between 3 and 6 individual zinc finger repeats and can recognize between 9 and 18 contiguous nucleotides. Each ZFN can be designed to target a specific target site in the host cell genome, e.g., a promotor sequence, an enhancer sequence, or exon/intron within a gene.

TALENs [0118] In some embodiments of the methods and compositions described herein, the nuclease is a TALEN. TAL effectors (TALEs) are proteins secreted by Xanthomonas bacteria and play an important role in disease or triggering defense mechanisms, by binding host DNA and activating effector-specific host genes. see, e.g., Gu et al. (2005) Nature 435:1122-5; Yang et al., (2006) Proc. Natl. Acad. Sci. USA 103:10503-8; Kay et al., (2007) Science 318:648-51; Sugio et al., (2007) Proc. Natl. Acad. Sci. USA 104:10720-5; Romer et al., (2007) Science 318:645-8; Boch et al., (2009) Science 326(5959):1509-12; and Moscou and Bogdanove, (2009) 326(5959):1501. A TALEN comprises a TAL effector DNA-binding domain fused to a DNA cleavage domain. The DNA binding domain interacts with DNA in a sequence-specific manner through one or more tandem repeat domains. The repeated sequence typically comprises 33-34 highly conserved amino acids with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD) are highly variable and show a strong correlation with specific nucleotide recognition (Boch et al., (2009) Science 326(5959):1509-12; and Moscou and Bogdanove, (2009) 326(5959):1501). This relationship between amino acid sequence and DNA recognition sequence has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing the appropriate RVDs. id="p-119"

[0119] The TAL-effector DNA binding domain can be engineered to bind to a target DNA sequence and fused to a nuclease domain, e.g., a Type IIS restriction endonuclease, such as FokI (see e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160). In some embodiments, the nuclease domain can comprise one or more mutations (e.g., FokI variants) that improve cleavage specificity (see, Doyon et al., (2011) Nature Methods, 8 (1): 74 –9) and cleavage activity (Guo et al., (2010) Journal of Molecular Biology, 400 (1): 96 –107). Other useful endonucleases that can be used as the nuclease domain include, but are not limited to, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. In some embodiments, the TALEN can comprise a TAL effector DNA binding domain comprising a plurality of TAL effector repeat sequences that bind to a specific nucleotide sequence (i.e., recognition sequence) in the target DNA. While not to be construed as limiting, TALENs useful for the methods provided herein include those described in WO10/079430 and U.S. Patent Application Publication No. 2011/0145940. id="p-120"

[0120] In some embodiments, the TAL effector DNA binding domain can comprise 10 or more DNA binding repeats, and preferably 15 or more DNA binding repeats. In some embodiments, each DNA binding repeat comprises a RVD that determines recognition of a base pair in the target DNA, and wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA. In some embodiments, the RVD comprises one or more of: HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, where * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, where * represents a gap in the second position of the RVD; IG for recognizing T; NK for recognizing G; HA for recognizing C; ND for recognizing C; HI for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; and YG for recognizing T. id="p-121"

[0121] In some embodiments, the TALEN is engineered such that the TAL effector comprises one or more transcriptional activator domains (e.g., VP16, VP48, VP64 or VP160). For example, engineered TAL effectors having a transcriptional activator domain at the c-terminus of the TAL effector were shown to modulate transcription of Sox2 and Klf4 genes in human 293FT cells (Zhang et al., Nature Biotechnology, 29(2):149-153 (2011). Other TAL effector transcription factors (TALE-TFs) are also known in the art, including those disclosed in Perez-Pinera et al., (Nature Methods, (2013) 10(3):239-242) that demonstrated modulation of IL1RN, KLK3, CEACAM5 and ERBB2 genes in human 293T cells using TALE-TFs. In some embodiments, the one or more transcriptional activator domains are located adjacent to the nuclear localization signal (NLS) present in the C-terminus of the TAL effector. In another embodiment, the TALE-TFs can bind nearby sites upstream or downstream of the transcriptional start site (TSS) for a target gene. In one embodiment, the TAL effector comprises a transcriptional activator domain selected from VP16, VP48, VP64 or VP160. In another embodiment, the TAL effector comprises a transcriptional activator domain selected from HSF1, VP16, VP64, p65, RTA, MyoD1, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9. id="p-122"

[0122] In some embodiments, the TALEN comprises a TAL effector DNA-binding domain fused to a DNA cleavage domain, wherein the TAL effector comprises a transcriptional activator. In some embodiments, the DNA cleavage domain is of a Type IIS restriction endonuclease selected from a HO endonuclease or a FokI endonuclease. In some embodiments, the TAL effector DNA-binding domain specifically binds to a recognition sequence corresponding to a promoter region or enhancer region disclosed herein (e.g., SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A and PAX6 promotor or enhancer).

H. Expression vectors for selected MHC alleles id="p-123"

[0123] In some embodiments, the engineered mammalian dendritic cell described herein contains one or more expression vectors for expressing the exogenous MHC class II alleles. A wide variety of expression vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons. Viral vectors that may be used, for example, include vectors based on HIV, SV40, EBV, HSV or BPV. The expression vectors may be replication-defective by design such that the viral vector is defective for one or more functions that are essential for viral genome replication or synthesis and assembly of viral particles. Many of the currently existing replication-defective viruses can carry large therapeutic genes, effectively transduce various types of cells, and provide long-term and stable expression of genes of interest. id="p-124"

[0124] Lentiviruses are a subset of retroviruses commonly used in research. Lentiviruses can transduce both dividing and non-dividing cells without a significant immune response. These viruses also integrate stably into the host genome, enabling long term transgene expression. A common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types. id="p-125"

[0125] One safety feature of lentiviruses is that the components necessary to produce an infectious viral particle (a virion) are generally divided among multiple plasmids. For instance, an infectious viral particle may comprise plasmids that components of the viral capsid and envelope (typically called the packaging and envelope plasmids), and plasmid that encodes the viral genome (typically called the transfer plasmid). Common lentiviral packaging and envelope plasmids that can be used herein include, but are not limited to, pRSV-Rev, pMDLg/pRRE, psPAX2, pCMV delta R8.2, pMD2.G, pCMV-VSV-G, pCMV-dR8.2 dvpr, pCI-VSVG, pCPRDEnv, pLTR-RD114A, pLTR-G, pCD/NL-BH*DDD, psPAX2-D64V, pCEP4-tat, pHEF-VSVG, pNHP, pCAG-Eco, and pCAG-VSVG. Common lentiviral transfer plasmids that can be used herein include, but are not limited to, pLKO.1 puro, pLKO.1 – TRC cloning plasmid, pLKO.3G, Tet-pLKO-puro, pSico, pLJM1-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLX301, plnducer20, pHIV-EGFP, Tet-pLKO-neo, pLV-mCherry, pCW57.1, pLionII, pSLIK-Hygro, and pInducer10-mir-RUP-PheS. id="p-126"

[0126] There are multiple approaches to produce lentiviral vectors. (See Logan et al. "Factors influencing the titer and infectivity of lentiviral vectors." Hum Gene Ther. 2004 Oct;15(10):976- 88. doi: 10.1089/hum.2004.15.976. PMID: 15585113; Dull, T et al. "A third-generation lentivirus vector with a conditional packaging system." Journal of virology vol. 72,11 (1998): 8463-71. doi:10.1128/JVI.72.11.8463-8471.1998). Alternatively, lentiviral vectors may be purchased from commercial providers. In general, production of lentiviral vectors involves multiple steps including plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, fill and finish. (See e.g., www.addgene.org/viral-vectors/lentivirus/; www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/lv-production-workflow.html). id="p-127"

[0127] The lentiviral vector may be designed to express one or more genes of interest simultaneously. Various molecular strategies are available, including the use of multiple promoters, signals of splicing, fusion of genes, cleavage factors and multicistronic vectors. (See e.g., review by Shaimardanova et al., "Production and application of multicistronic constructs for various human disease therapies." Pharmaceutics 2019, 11, 580.) id="p-128"

[0128] In some embodiments, the engineered mammalian dendritic cells described herein are expressed using non-viral approaches. Exemplary methods include, but are not limited to, cationic lipids such as liposomes and lipoplexes, polymers or polyplexes and dendrimers, naked plasmids for direct delivery, electroporation, ultrasound and micro bubbles, magnetofections, inorganic molecules.

I. Compositions id="p-129"

[0129] In one aspect, the present disclosure provides a composition comprising an engineered mammalian dendritic cell. As described herein, the engineered mammalian dendritic cell comprises one or more exogenous alleles of an MHC class II gene. id="p-130"

[0130] In some embodiments, the composition comprises at least 10,000 cells, at least 100,0cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,0cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,0cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,0cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells. In some embodiments, the composition comprises at least 1,000,000 cells. In some embodiments, the composition comprises at least 20,000,000 cells. id="p-131"

[0131] In some embodiments, the composition comprises at most 10,000 cells, at most 100,0cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,0cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,0cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,0cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,000 cells, or at most 50,000,000 cells. In some embodiments, the composition comprises at most 20,000,0cells. In some embodiments, the composition comprises at most 40,000,000 cells. id="p-132"

[0132] In some embodiments, the composition comprises about 1,000,000 to about 50,000,0cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the composition comprises about 1,000,000 cells. In some embodiments, the composition comprises about 20,000,000 cells. In some embodiments, the composition comprises about 40,000,000 cells. id="p-133"

[0133] In another aspect, the present disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the compositions described herein and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition may comprise an engineered mammalian dendritic cell or cell line comprising at least 1, 2, 3, 4, 5, or more exogenous alleles encoding at least one MHC class II gene. In some embodiments, the engineered mammalian dendritic cell or cell line may also comprise at least 1, 2, 3, 4, 5, or more exogenous alleles encoding at least one MHC class I gene. In some embodiments, the engineered mammalian dendritic cell or cell line may further comprise one or more co-stimulatory molecules, heterologous antigens, and/or cytokine described herein. The at least 1, 2, 3, 4, 5, or more recombinant polynucleotides may comprise a heterologous sequence encoding, e.g., a co-stimulatory molecule, a heterologous antigen, a cytokine, or a 2A splicing peptide. Thus, the recombinant polynucleotide encoding the one or more MHC alleles, co-stimulatory molecule, heterologous antigen, and/or cytokine may be separated by a sequencing encoding a 2A splicing peptide (e.g., T2A, P2A, E2A). Generally, at least 1, 2, 3, 4, 5, or more exogenous alleles are cloned into an expression vector (e.g., a replication defective lentiviral vector) for synthesis of the MHC alleles, co-stimulatory molecule, heterologous antigen, and/or cytokine, and introduced into the engineered mammalian dendritic cell or cell line. Accordingly, the engineered mammalian dendritic cell or cell line provided in the pharmaceutical composition may have at least 1, 2, 3, 4, , or more expression vectors, each comprising at least 1, 2, 3, 4, 5 or more exogenous alleles encoding the MHC alleles, co-stimulatory molecule, heterologous antigen, and/or cytokine. id="p-134"

[0134] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant, an interferon alpha (e.g., IFN-α2a or IFN-α2b), and/or an interferon lambda family member (e.g., an interferon lambda 1 (IFNλ1 (IL-29)), an interferon lambda 2 (IFNλ2 (IL-28A)), an interferon lambda 3 (IFNλ1 (IL-28B)), an interferon lambda 4 (IFNλ4)). In some embodiments, the interferon alpha (e.g., IFN-α2a or IFN-α2b) is expressed by a vector comprising a polynucleotide sequence of the IFNA2 gene in the engineered mammalian dendritic cell as described herein. In some embodiments, the interferon alpha is a pegylated IFN-α2a provided exogenously. In some embodiments, the pharmaceutical composition further comprises one or more excipients. In some embodiments, the pharmaceutical composition further comprises CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation media. In certain embodiments, the pharmaceutical composition comprises cells cryopreserved in CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation media. id="p-135"

[0135] In some embodiments, the pharmaceutical composition is formulated in a dosage form comprising a total number of engineered mammalian dendritic cell per dose for administration to a subject in need therefor. In some embodiments, the pharmaceutical composition is formulated as an "off-the-shelf" product for self-administration to a subject in need thereof. In some embodiments, the pharmaceutical composition may have at least at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 20,000,000 cells. id="p-136"

[0136] In some embodiments, the pharmaceutical composition comprises at most 10,000 cells, at most 100,000 cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,000 cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,000 cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,000 cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,0cells, or at most 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 40,000,000 cells. id="p-137"

[0137] In some embodiments, the pharmaceutical composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 40,000,000 cells. id="p-138"

[0138] In some embodiments, the pharmaceutical composition is formulated in the form of a suspension. The formulation of pharmaceutical compositions is generally known in the art (see, e.g., REMINGTON ’S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)). Prevention against microorganism contamination can be achieved through the addition of one or more of various antibacterial and antifungal agents. In certain embodiments, the pharmaceutical composition is a liquid formulation comprising cells resuspended in Lactated Ringer’s solution. id="p-139"

[0139] Pharmaceutical forms suitable for administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Typical carriers include a solvent or dispersion medium containing, for example, water-buffered aqueous solutions (i.e., biocompatible buffers, non-limiting examples of which include Lactated Ringer’s solution and CryoStor cryopreservation media (e.g., CS2, CS5, and CS10, containing 2%, 5%, and 10%, respectively of DMSO; available from BioLife Solutions, Bothell, WA)), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils. id="p-140"

[0140] Sterilization can be accomplished by an art-recognized technique, including but not limited to addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions. id="p-141"

[0141] Production of sterile injectable solutions containing engineered mammalian dendritic cell(s), and/or other composition(s) of the present disclosure can be accomplished by incorporating the compound(s) in the required amount(s) in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization. To obtain a sterile powder, the above sterile solutions can be vacuum-dried or freeze-dried as necessary. id="p-142"

[0142] In some embodiments, the engineered mammalian dendritic cell(s), and/or other composition(s) provided herein are formulated for administration, e.g., intradermal injection, intralymphatic injection, oral, nasal, topical, or parental administration in unit dosage form for ease of administration and uniformity of dosage. Unit dosage forms, as used herein, refers to physically discrete units suited as unitary dosages for the subjects, e.g., humans or other mammals to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some instances, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the engineered mammalian dendritic cell(s), and/or other composition(s). id="p-143"

[0143] In some embodiments, the engineered mammalian dendritic cell(s), and/or other composition(s) provided herein are formulated for administration e.g., one or more doses over a period of time. In some embodiments, the engineered mammalian dendritic cell(s), and/or other composition(s) are formulated for administration every week, every 2 weeks, every 3 weeks, every weeks, every 5 weeks, or every 6 weeks. In some embodiments, the engineered mammalian dendritic cell(s), and/or other composition(s) are formulated for administration every month, every months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months. id="p-144"

[0144] A dose may include, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, or more) engineered mammalian dendritic cells. In some embodiments, a dose may contain about 1,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 5,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 10,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 20,000,000 engineered mammalian dendritic cells. id="p-145"

[0145] A dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) engineered mammalian dendritic cells. In some embodiments, a dose may include at least about 1,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may include at least about 5,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may include at least about 10,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may include at least about 20,000,0engineered mammalian dendritic cells. id="p-146"

[0146] A dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000, 650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000, 950,000,000, 1,000,000,000, or more) engineered mammalian dendritic cells. id="p-147"

[0147] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON ’S PHARMACEUTICAL SCIENCES, supra). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON ’S PHARMACEUTICAL SCIENCES, supra). id="p-148"

[0148] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes. id="p-149"

[0149] In some embodiments, the therapeutically effective dose may further comprise other components, for example, anti-allergy drugs, such as antihistamines, steroids, bronchodilators, leukotriene stabilizers and mast cell stabilizers. Suitable anti-allergy drugs are well known in the art.

J. Methods for Treating Cancer Methods for semi-allogeneic dendritic cell-based immunotherapy [0150] The present disclosure provides a method for semi-allogeneic dendritic cell-based immunotherapy in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the present disclosure) described herein. id="p-151"

[0151] In some embodiments, the method comprises, prior to the administering, (i) obtaining an MHC class II allele profile by genotyping a plurality of MHC class II genes in a biological sample from the subject; and (ii) selecting an engineered mammalian dendritic cell for administering to the subject, wherein the engineered mammalian dendritic cell comprises one or more mismatches to the MHC class II allele profile of the subject. In some embodiments, the subject is a human. In some embodiments, the method for semi-allogeneic dendritic cell-based immunotherapy is for treating a cancer in a human. In some instances, the engineered human dendritic cell or a plurality thereof for the cancer treatment comprises a tumor-specific antigen or a fragment thereof.

Methods for autologous dendritic cell-based immunotherapy [0152] The present disclosure also provides a method for autologous dendritic cell-based immunotherapy in a subject. As described herein, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the present disclosure), wherein the engineered mammalian dendritic cell is derived from a primary cell of the subject. In some embodiments, the primary cell is a primary immune cell, including but not limit to, a dendritic cell, a monocyte, a macrophage, B cell, a T cell, a nature killer (NK) cell, and a neutrophil. A particular example of the primary immune cell is dendritic cell. id="p-153"

[0153] In some embodiments, the method for autologous dendritic cell-based immunotherapy further comprises, prior to the administering, (i) obtaining the primary immune cell or a plurality from the subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cell to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cell into an engineered mammalian dendritic cell by (a) introducing into the primary immune cells one or more exogenous MHC class II alleles comprising at least one mismatch to the endogenous MHC class II allele profile of the subject; and (b) introducing into the primary immune cell an antigen of the cancer or the pathogen the subject is suffering or a fragment thereof. In some embodiments, the engineering of the mammalian dendritic cells further comprises a step of: (c) incubating the primary immune cell with Lipopolysaccharide (LPS), interferon-gamma (IFN-γ), or a combination of LPS and IFN-γ.

Treg inhibitory agents [0154] In some embodiments, the disclosure provides a method for enhancing or inducing anti-cancer response in a subject via using regulatory T cell inhibitory agents (Treg inhibitory agents, or Treg agents) that inhibit or decrease the activity or function of Tregs in order to promote the efficacy of the cancer vaccines described herein. The method comprises administering to a subject an effective amount of a Treg agent to a subject, wherein the Treg agent decreases the activity or function of Tregs, and administering an effective amount of the pharmaceutical composition comprising engineered mammalian dendritic cells disclosed herein. In some instances, the Treg agent and the pharmaceutical composition are administered to the subject at the same time. In other instances, the Treg agent is administered to the subject before administering the pharmaceutical composition. In yet other instances, the Treg agent is administered to the subject after administering the pharmaceutical composition. In particular embodiments, the Treg agent is administered to the subject an hour, a few hours, a day, a few days, a week, a few weeks, a month, or a few months, after administering the pharmaceutical composition. id="p-155"

[0155] The regulatory T cell inhibitory agent (Treg inhibitory agent, or Treg agent) as disclosed herein, can be any Treg-targeting agent, including but not limited to, any compound, small molecule, toxin, polynucleotide (such as aptamer, RNAi, siRNA, or antisense oligonucleotide), polypeptide, protein (such as antibodies), fusion protein, drug conjugate, chemotherapeutic agents and the like, that (i) inhibits or decreases Treg cell functions, and/or (ii) depletes or diminishes regulatory T cell populations. id="p-156"

[0156] In some embodiments, the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA, a siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof. Examples of Treg agents include, but are not limited to, as those described in Yang, J., Bae, H. Drug conjugates for targeting regulatory T cells in the tumor microenvironment: guided missiles for cancer treatment. Exp Mol Med 55, 1996–2004, Kumar P, Kumar A, Parveen S, Murphy JR, Bishai W. Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy. Immunotherapy. 2019;11(13):1117-1128, Verma A, Mathur R, Farooque A, Kaul V, Gupta S, Dwarakanath BS. T-Regulatory Cells In Tumor Progression And Therapy. Cancer Manag Res. 2019;11:10731-10747, the entire disclosures of which are herein incorporated by reference. id="p-157"

[0157] In certain embodiments, a Treg agent can comprise an antibody, or a fragment thereof, which specifically binds to a regulatory T cell surface protein. Antibodies that comprise a Treg agent can target a surface protein of the Treg cell, which include, for example, CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), cytotoxic T lymphocyte-associated antigen (CTLA4), CCR4, CCR8, FOXP3, LAG3, CD103, NRP-1, glucocorticoid-induced TNF receptor (GITR), galectin-1, TNFR2, or TGF-βR1. In some embodiments, the Treg agent can be, for example, ONTAK, HuMax-Tac, Zenapax, or MDX-010 or a combination thereof. ONTAK is a monoclonal antibody that binds to the CD25 subunit of the IL-2 receptor. HuMax-TAC is a fully human monoclonal antibody that targets the TAC antigen. TAC is also known as CD25 or the alpha subunit of the interleukin-2 receptor (IL-2Rα) and is overexpressed by activated T-cells. Zenapax is an immunosuppressive, humanized IgG1 monoclonal antibody that binds to the CDsubunit of the human high-affinity IL-2 receptor expressed on the surface of activated lymphocytes. MDX-010 is a monoclonal antibody directed against CTLA4. id="p-158"

[0158] In certain embodiments, the Treg agent can comprise an antibody-drug conjugate. As disclosed herein, the antibody, or fragment thereof, can further comprise a radionuclide or toxic moiety such that the antibody can kill the regulatory T cell. In some embodiments, radionuclides suitable for use in the present disclosure can include those having suitable emission properties to provide ablation of targeted Tregs in situ, while not unduly exposing the surrounding cells and tissues to damaging levels of irradiation. An ideal radionuclide for use in such therapeutic compositions is a relatively short-lived α-emitter, a γ-emitter, or a β-emitter that emits enough gamma irradiation to cause local destruction. Non-limiting examples of radionuclides include lutetium-177, iodine-131, iodine-125, and phosphorus-32 (γ-emitters); actinium-225, astatine-211, and bismuth-212 and bismuth-213 (α-emitters); iodine-123, copper-64, iridium-192, osmium-194, rhodium-105, rhodium-186, samarium-153, and yttrium-88, yttrium-90, and yttrium-91. id="p-159"

[0159] In certain embodiments, the Treg agent can comprise a fusion protein. In some embodiments, the fusion protein can comprise a targeting moiety and a toxic moiety. The targeting moiety can comprise a ligand or portion thereof of a regulatory T cell surface protein. The ligand can be, for example, IL2, T cell receptor (TCR), MHCII, CD80, CD86, TARC, CCL17, CKLF1, CCL1, TCA-3, eotaxin, TER-1, E-cadherin, VEGF, semaphorin3a, CD134, CD31, CD62, CD38L, or glucocorticoid-induced TNF receptor ligand (GITRL). The toxic moiety can comprise, for example, lectin, ricin, abrin, viscumin, modecin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, botulinum toxin, tetanus toxin, calicheamicin, or pokeweed antiviral protein. id="p-160"

[0160] In certain embodiments, the Treg agent can comprise an immunotoxin. An immunotoxin can be an immunoconjugate that induces death of a target cell by combining an antibody with target-specific high-affinity binding activity with other molecules, such as radioisotopes, chemicals, siRNA, and cytotoxic proteins. In some embodiments, the immunotoxin comprises Denileukin diftitox (Ontak), Tagraxofusp (Elzonris), Moxetumomab pasudotox (Lumoxiti), or any derivative, combination thereof. id="p-161"

[0161] In certain embodiments, the Treg agent can comprise a peptide–drug conjugate (PDC). A PDC comprises a peptide linked to a payload. The peptide in the PDC can be target cell specific and induce receptor-mediated endocytosis of the conjugate. The payload can be highly toxic drug such as maytansine, camptothecin derivatives, auristatin, or doxorubicin. id="p-162"

[0162] In certain embodiments, the Treg agent can comprise a small interfering RNA (siRNA). siRNA is a double-stranded RNA (dsRNA) molecule 21–23 nucleotides in length that specifically causes RNA interference (RNAi), a posttranscriptional method of silencing gene expression. In certain embodiments, the siRNA conjugates with a biomolecule, such as a lipophilic molecule, an antibody, an aptamer, a ligand, a peptide, or a polymer. Aptamers are single-stranded oligonucleotides that recognize their targets through their unique three-dimensional complementarity. In some embodiments, the Treg agent can comprise an siRNA conjugate, such as CTLA4apt-STAT3 siRNA, NPsiCTLA-4, or a hybrid SNP. CTLA4apt-STAT3 siRNA is a siRNA conjugate in which CTLA4 binding RNA aptamer and mouse STAT3 siRNA are linked. NPsiCTLA-4 is a nanostructure material-siRNA conjugate in which siRNA-targeting CTLA-mRNA is surrounded by nanoparticles composed of PEG5k–PLA11k and BHEM-Chol. Hybrid SNPs are spherical nucleotide nanoparticles (SNPs) loaded with a CTLA-4-siRNA aptamer (cSNP) and PD-1 siRNA (pSNP) in a nanoparticle comprising an amphiphilic polymer of PLGA-S-S-PEG as the core and the cationic lipid DOTAP. id="p-163"

[0163] According to the methods of the disclosure, a Treg agent modulates a regulatory T cell via either decreasing the activity or function of a Treg after the Treg agent is administered to a subject; or a Treg agent that is attached to a toxic moiety can kill or ablate regulatory T cells. The administration of a Treg agent or derivatives thereof can block the action of its target (for example a Treg cell surface marker). Thus, a Treg agent can decrease the suppression of immune system activation and can decrease the prevention of self-reactivity. Such a decrease can be measured via techniques established in the art. For example, see Dannull et al., (2005) J Clin Invest 115(12):3623-33; and Tsaknaridis, et al., (2003) J Neurosci Res 74: 296-308. Non-limiting examples of assays used for the detection of T cell responses include delayed-type hypersensitivity responses; in vitro T cell proliferation responses (e.g., measured by incorporation of radioactive nucleotides); library screens; expression arrays; T cell cytokine responses (e.g., measured by ELISA or other related immuno-assays or RT-PCR for specific cytokine mRNA); as well as any other assay established in the art for measuring a B cell and/or T cell immune response in a subject. Methods for detecting an immune response can include, but are not limited to, antibody detection assays such as, for example, EIA (enzyme immunoassay); ELISA (enzyme linked immunosorbent assay); agglutination reactions; precipitation/flocculation reactions, immunoblots (Western blot; dot/slot blot); radioimmunoassays; immunodiffusion assays (RIA); histochemical assays; immunofluorescence assays (FACS); chemiluminescence assays, library screens, expression arrays, etc.

In combination with other agents or therapies [0164] In some embodiments, the method further comprising administering to the subject one or more doses of cyclophosphamide intravenously at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or longer, prior to administering to the subject the pharmaceutical composition described herein. In some embodiments, the cyclophosphamide is administered at least about 2-3 days prior to administering to the subject the pharmaceutical composition described herein. In some embodiments, a low-dose of cyclophosphamide at about 100, 150, 200, 250, 300, or 450 mg/m is administered to the subject. id="p-165"

[0165] In some embodiments, the method further comprising administering to the subject one or more doses of an interferon-alpha-2b (IFN-α2b), IFN-α2a, or a pegylated IFN-α2a intradermally at the inoculation site of the pharmaceutical composition described herein. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours following administering to the subject the pharmaceutical composition described herein. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally at about 1-4 hours, about 2-hours, about 8-12 hours, about 10-24 hours, about 20-48 hours, or about 60-72 hours following administering to the subject the pharmaceutical composition described herein. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally no later than 5, 10, 15, 20, 25, 30, 45, 50, 60, 72, or 84 hours after administering to the subject the pharmaceutical composition. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally no later than 1, 2, 3, 4, 5, or 6 days following administering to the subject the pharmaceutical composition. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally no later than about 1-6 days, 2-3 days, or 3-5 days following administering to the subject the pharmaceutical composition. In some embodiments, the method further comprising administering to the subject a first doses of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally between 1 to 4 hours and a second dose of IFN-α2b, IFN-α2a or pegylated IFN-α2a intradermally between 1-3 days following administering to the subject the pharmaceutical composition. In some embodiments, the IFN-α2b, administered is at a low-dose between about 1-20,000 IU, 100-15,000 IU, 5000-12,000 IU, or 9,000-11,000 IU. In some embodiments, the IFN-α2b administered is at dose of about 10,000 IU. In some embodiments, the IFN-α2a or pegylated IFN-α2a, administered is at a low-dose between about 0.01-0.1 micrograms (mcg), 0.05 – 0.mcg, 0.06 – 0.12 mcg, or 0.09 -0.11 mcg. In some embodiments, the IFN-α2b administered is at dose of about 0.1 mcg. id="p-166"

[0166] In some embodiments, the method further comprises administering to the subject one or more additional therapies. Examples of suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiotherapy, hormone therapy, a differentiating agent, and a small-molecule drug. One of skill in the art will readily be able to select an appropriate additional therapy. id="p-167"

[0167] Chemotherapeutic agents that can be used in the present disclosure include but are not limited to alkylating agents (e.g., nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozocin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), triazines (e.g., dacarbazine (DTIC), temozlomide), ethylenimines (e.g., thiotepa, altretamine (hexamethylmelamine)), platinum drugs (e.g., cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed), anthracycline anti-tumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline anti-tumor antibiotics (e.g., actinomycin-D, bleomycin, mitomycin-C, mitoxantrone), mitotic inhibitors (e.g., taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine), estramustine, corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), L-asparaginase, bortezomib, and topoisomerase inhibitors. Combinations of chemotherapeutic agents can be used. id="p-168"

[0168] Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerases, which are enzymes that facilitate changes in DNA structure by catalyzing the breaking and rejoining of phosphodiester bonds in the backbones of DNA strands. Such changes in DNA structure are necessary for DNA replication during the normal cell cycle. Topoisomerase inhibitors inhibit DNA ligation during the cell cycle, leading to an increased number of single- and double-stranded breaks and thus a degradation of genomic stability. Such a degradation of genomic stability leads to apoptosis and cell death. id="p-169"

[0169] Topoisomerases are often divided into type I and type II topoisomerases. Type I topoisomerases are essential for the relaxation of DNA supercoiling during DNA replication and transcription. Type I topoisomerases generate DNA single-strand breaks and also religate said breaks to re-establish an intact duplex DNA molecule. Examples of inhibitors of topoisomerase type I include irinotecan, topotecan, camptothecin, and lamellarin D, which all target type IB topoisomerases. id="p-170"

[0170] Type II topoisomerase inhibitors are broadly classified as topoisomerase poisons and topoisomerase inhibitors. Topoisomerase poisons target topoisomerase-DNA complexes, while topoisomerase inhibitors disrupt enzyme catalytic turnover. Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones. id="p-171"

[0171] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some instances, the topoisomerase inhibitor is a topoisomerase I inhibitor, a topoisomerase II inhibitor, or a combination thereof. In certain embodiments, the topoisomerase inhibitor is selected from the group consisting of doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, an ellipticine, aurintricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, lamellarin D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), and a combination thereof. EGCG is one example of a plant-derived natural phenol that serves as a suitable topoisomerase inhibitor. In some instances, the topoisomerase inhibitor is doxorubicin. id="p-172"

[0172] Immunotherapy refers to any treatment that uses the subject’s immune system to fight a disease (e.g., cancer). Immunotherapy methods can be directed to either enhancing or suppressing immune function. In the context of cancer therapies, immunotherapy methods are typically directed to enhancing or activating immune function. In some instances, an immunotherapeutic agent comprises a monoclonal antibody that targets a particular type or part of a cancer cell. In some cases, the antibody is conjugated to a moiety such as a drug molecule or a radioactive substance. Antibodies can be derived from mouse, chimeric, or humanized, as non-limiting examples. Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, retifanlimab, rituximab, tositumomab, and trastuzumab. id="p-173"

[0173] Immunotherapeutic agents can also comprise an immune checkpoint inhibitor, which modulates the ability of the immune system to distinguish between normal and "foreign" cells. Programmed cell death protein 1 (PD-1) and protein death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors, as disruption of the interaction between PD1 and PD-L1 enhance the activity of immune cells against foreign cells such as cancer cells. Examples of PD-1 inhibitors include pembrolizumab, retifanlimab and nivolumab. An example of a PD-L1 inhibitor is atezolizumab. id="p-174"

[0174] Another immune checkpoint target for the treatment of cancer is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which is a receptor that downregulates immune cell responses. Therefore, drugs that inhibit CTLA-4 can increase immune function. An example of such a drug is ipilimumab, which is a monoclonal antibody that binds to and inhibits CTLA-4. id="p-175"

[0175] The term "radiotherapy" refers to the delivery of high-energy radiation to a subject for the treatment of a disease (e.g., cancer). Radiotherapy can comprise the delivery of X-rays, gamma rays, and/or charged particles. Radiotherapy can be delivered locally (e.g. to the site or region of a tumor), or systemically (e.g., a radioactive substance such as radioactive iodine is administered systemically and travels to the site of the tumor). id="p-176"

[0176] The term "hormone therapy" can refer to an inhibitor of hormone synthesis, a hormone receptor antagonist, or a hormone supplement agent. Inhibitors of hormone synthesis include but are not limited to aromatase inhibitors and gonadotropin releasing hormone (GnRH) analogs. Hormone receptor antagonists include but are not limited to selective receptor antagonists and antiandrogen drugs. Hormone supplement agents include but are not limited to progestogens, androgens, estrogens, and somatostatin analogs. Aromatase inhibitors are used, for example, to treat breast cancer. Non-limiting examples include letrozole, anastrozole, and aminoglutethimide. GnRH analogs can be used, for example, to induce chemical castration. Selective estrogen receptor antagonists, which are commonly used for the treatment of breast cancer, include tamoxifen, raloxifene, toremifene, and fulvestrant. Antiandrogen drugs, which bind to and inhibit the androgen receptor, are commonly used to inhibit the growth and survival effects of testosterone on prostate cancer. Non-limiting examples include flutamide, apalutamide, and bicalutamide. id="p-177"

[0177] The term "differentiating agent" refers to any substance that promotes cell differentiation, which in the context of cancer can promote malignant cells to assume a less stem cell-like state. A non-limiting example of an anti-cancer differentiating agent is retinoic acid. id="p-178"

[0178] Small molecule drugs generally are pharmacological agents that have a low molecular weight (i.e., less than about 900 daltons). Non-limiting examples of small molecule drugs used to treat cancer include bortezomib (a proteasome inhibitor), imatinib (a tyrosine kinase inhibitor), and seliciclib (a cyclin-dependent kinase inhibitor), and epacadostat (an indoleamine 2,3-dioxygenase (IDO1) inhibitor).

Administration [0179] In some embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition intradermally in the upper back or thighs. Without being bound by any theories, the upper back and thighs are chosen for patient acceptability as these areas have less nerves in the skin and are thus less sensitive. Additionally, the draining lymph nodes in the proximity may convey antigens from breast tumors in the upper and lower torso, which are common sites for breast cancer metastases. The method may further comprise administering to the subject the pharmaceutical composition in an interval of every week, every weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the method comprises administering to be subject the pharmaceutical composition in an interval of every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 6 weeks, weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 1 month, months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, or 52 weeks. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, months, 10 months, or 12 months. id="p-180"

[0180] In some embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition through oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration. In some embodiments, administration of the effective amount of the pharmaceutical composition is performed by parenteral administration (e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial) or transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). In some embodiments, the method comprises the use of liposomal formulations, intravenous infusion, or transdermal patches. id="p-181"

[0181] Therapy such as engineered mammalian dendritic cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered using routes, dosages, and protocols that will readily be known to one of skill in the art. Administration can be conducted once per day, once every two days, once every three days, once every four days, once every five days, once every six days, or once per week. Therapy can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more times per week. In some cases, engineered mammalian dendritic cell(s), composition(s), and/or pharmaceutical composition(s) of the present disclosure are administered as a single dose, co-administered (e.g., administered in separate doses or by different routes, but close together in time), or administered separately (e.g., administered in different doses, including the same or different route, but separated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours). In cases where multiple doses are to be administered in the same day, or where a single dose comprises one or more components (e.g., the engineered mammalian dendritic cell(s) and IFNa are administered separately), administration can occur, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times in a day. id="p-182"

[0182] In some cases, therapeutic administration can occur about once per week, about every two weeks, about every three weeks, or about once per month. In other cases, therapeutic administration can occur about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more times per month. Treatment can continue for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; or longer. At any time during treatment, the therapeutic plan can be adjusted as necessary. For example, depending on the response to engineered mammalian dendritic cell(s), compositions, or pharmaceutical composition(s) of the present disclosure, a different vaccine may be selected, one or more additional therapeutic agents or drugs may be chosen, or any aspect of the therapeutic plan can be discontinued. One of skill in the art will readily be able to make such decisions, which can be informed by, for example, the results of allele profile comparison, changes in the activity and/or number of an immune cell, and/or changes in the the presence or level of one or more biomarkers. id="p-183"

[0183] The engineered mammalian dendritic cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered by any suitable route, including those described herein. In some embodiments, the administration is by intradermal or intralymphatic injection. In some embodiments, the whole-cell cancer vaccine (e.g., comprising engineered mammalian dendritic cells of the present disclosure) is given separately from interferon alpha (IFNa). In some instances, the IFNa is injected locally. IFNa can be given before and/or after the vaccine is administered. Timing of the separate injections can be any suitable interval, including those described herein. id="p-184"

[0184] One of skill in the art will readily be able to administer the number of appropriate engineered mammalian dendritic cells to include in a particular dose. A dose may include, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, or more) engineered mammalian dendritic cells. In some embodiments a dose may contain about 1,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 5,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 10,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 20,000,000 engineered mammalian dendritic cells. id="p-185"

[0185] A dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) engineered mammalian dendritic cells. id="p-186"

[0186] A dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000, 650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000, 950,000,000, 1,000,000,000, or more) engineered mammalian dendritic cells. id="p-187"

[0187] In some embodiments, the engineered mammalian dendritic cells are irradiated. The irradiation dose may be, for example, between about 2 and 2,000 Gy (e.g., about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Gy). In certain embodiments, the engineered mammalian dendritic cells are irradiated with a dose of about 100 Gy.

Vaccine effects [0188] In some embodiments, the method of treating cancer of the present disclosure further comprises selecting a whole-cell cancer vaccine for the subject according to a method of the present disclosure described herein. In certain embodiments, the subject has stage I, stage II, stage III, and/or stage IV cancer. In other embodiments, the cancer is transitioning between stages. In some embodiments, the subject has a pre-cancerous lesion. In some embodiments, the subject does not have cancer. id="p-189"

[0189] In some embodiments, treating the subject comprises inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, ameliorating or eliminating the symptoms of cancer, reducing the size (e.g., volume) of a cancer tumor, reducing the number of cancer tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effects of a composition or pharmaceutical composition. In some embodiments, treating the subject results in an increased survival time. In some instances, overall survival is increased. In other instances, disease-free survival is increased. In some instances, progression-free survival is increased. In certain embodiments, treating the subject results in a reduction in tumor volume and/or increased survival time. id="p-190"

[0190] In certain embodiments, treating the subject enhances the therapeutic effects of an anti-cancer therapy such as a chemotherapeutic agent, an immunotherapeutic agent, radiotherapy, hormone therapy, a differentiating agent, and/or a small-molecule drug. id="p-191"

[0191] In some embodiments, treating the subject results in a decrease in the presence or level of one or more heterologous antigens measured or detected in a sample obtained from the subject. In some embodiments, treating the subject results in an increase in the presence or level of one or more biomarkers measured or detected in a sample obtained from the subject. In certain embodiments, treating the subject results in no change the presence or level of the one or more biomarkers. id="p-192"

[0192] In some embodiments, treating the subject results in an increase in the activity and/or number of one or more immune cells. In some instances, the increase is produced in one cell type. In other instances, the increase is produced in multiple cell types. In some embodiments, the cell in which the level of activity and/or number is increased is selected from the group consisting of a peripheral blood mononuclear cell (PBMC), a lymphocyte (e.g. T lymphocyte, B lymphocyte, NK cell), a monocyte, a dendritic cell, a macrophage, a myeloid-derived suppressor cell (MDSC), and a combination thereof. In certain embodiments, the level of activity and/or number of immune cell(s) is measured using methods of the present disclosure described herein. id="p-193"

[0193] In some embodiments, an increase in immune cell activity and/or number indicates that the subject should be administered one or more additional doses of the pharmaceutical composition (e.g., comprising engineered mammalian dendritic cells of the present disclosure). In some instances, a different vaccine is administered. One of skill in the art will recognize that an increase in immune cell activity and/or number will occur, in some instances, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or more doses of the vaccine have been administered. id="p-194"

[0194] In some embodiments, a sample is obtained from the subject. In other embodiments, a sample is obtained from a different subject or a population of subjects. Samples can be used for the purposes of selecting an appropriate cancer vaccine of the present disclosure, monitoring the response to vaccine therapy, and/or predicting how the subject will respond to vaccine therapy. Samples obtained from a different subject and/or a population of subjects can be used, for example, to establish reference ranges to facilitate comparisons that are part of the methods of the present disclosure. Samples can be obtained at any time, including before and/or after administration of the engineered mammalian dendritic cell(s), pharmaceutical composition(s), and/or other composition(s) of the present disclosure. In some embodiments, the sample comprises whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal cells, tumor tissue, urine, fluid obtained from a pleural effusion, hair, skin, or a combination thereof. In general, the sample can comprise any biofluid. For MHC typing, any cell, tissue, or biofluid type is suitable, as long as it contains a sufficient amount of DNA or RNA to allow typing. In some instances, the sample comprises circulating tumor cells (CTCs). The sample can also be made up of a combination of normal and cancer cells. In certain embodiments, the sample comprises circulating tumor cells (CTCs). The sample can be obtained, for example, from a biopsy, from a surgical resection, and/or as a fine needle aspirate (FNA). Samples can be used to determine, measure, or detect MHC allele(s), immune cell activity and/or number, and/or biomarker(s), as described herein. id="p-195"

[0195] In some embodiments, the results of the MHC typing (e.g., the alleles present in an allele profile, the results of a comparison of allele profiles), immune cell activity and/or number measurement, and/or biomarker presence or level determinations are recorded in a tangible medium. For example, the results of assays (e.g., the alleles present in an allele profile, the results of a comparison of allele profiles, the activity level and/or number of immune cells, the presence or level (e.g., expression) of one or more biomarkers and/or a prognosis or diagnosis (e.g., of whether or not there is the presence of cancer, the prediction of whether the subject will respond to a vaccine, or whether the subject is responding to a vaccine) can be recorded, e.g., on paper or on electronic media (e.g., audio tape, a computer disk, a CD, a flash drive, etc.). id="p-196"

[0196] In other embodiments, the methods further comprise the step of providing the results of assays, prognosis, and/or diagnosis to the patient (i.e., the subject) and/or the results of treatment.

K. Kits id="p-197"

[0197] In another aspect, the present disclosure provides a kit for treating a subject with a cancer. In some embodiments, the kit comprises an engineered mammalian dendritic cell line, a composition, and/or a pharmaceutical composition of the present disclosure described herein. The kits are useful for treating any cancer, some non-limiting examples of which include breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, liver cancer, brain cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including a combination thereof. id="p-198"

[0198] Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods. As used herein, the term "kit" includes a combination of articles that facilitates a process, assay, analysis, or manipulation. In particular, the kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like. id="p-199"

[0199] Kits can contain chemical reagents as well as other components. In addition, the kits of the present disclosure can include, without limitation, instructions to the kit user, apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, apparatus and reagents for administering engineered mammalian dendritic cell(s) or other composition(s) of the present disclosure, apparatus and reagents for determining the level(s) of biomarker(s) and/or the activity and/or number of immune cells, apparatus and reagents for detecting MHC alleles, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Kits of the present disclosure can also be packaged for convenient storage and safe shipping, for example, in a box having a lid. For instance, the kits may be stored and shipped at room temperature, on wet ice or with cold packs, or frozen in the vapor phase of liquid nitrogen or in dry ice. id="p-200"

[0200] In some embodiments, the kits also contain negative and positive control samples for detection of MHC alleles, immune cell activity and/or number, and/or the presence or level of biomarkers. In some embodiments, the negative control samples are non-cancer cells, tissue, or biofluid obtained from the subject who is to be treated or is already undergoing treatment. In other embodiments, the negative control samples are obtained from individuals or groups of individuals who do not have cancer. In other embodiments, the positive control samples are obtained from the subject, or other individuals or groups of individuals, who have cancer. In some embodiments, the kits contain samples for the preparation of a titrated curve of one or more biomarkers in a sample, to assist in the evaluation of quantified levels of the activity and/or number of one or more immune cells and/or biomarkers in a biological sample.

IV. Examples id="p-201"

[0201] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1: Mouse Strains and Dendritic Cells id="p-202"

[0202] This example illustrates how to generate syngeneic or semi-allogeneic bone marrow dendritic cells (BMDCs) from three mouse strains. id="p-203"

[0203] Each laboratory mouse strain is homozygous and has a unique MHC haplotype. In this study, three mouse strains were used to generate bone marrow dendritic cells (BMDCs): C57BL/6J (WT); B6.C-H2-Kbm1/ByJ (bm1); and B6(C)-H2-Ab1bm12/KhEgJ (bm12) ( FIGS. 1A-1C ). Both B6.C-H2-Kbm1/ByJ (bm1) and B6(C)-H2-Ab1bm12/KhEgJ (bm12) mouse strains are derived originally from C57BL/6J (WT) mouse strain. The B6.C-H2-Kbm1/ByJ (bm1) mouse strain has point mutations in the MHC class I H2-Kb allele. The B6(C)-H2-Ab1bm12/KhEgJ (bm12) mouse strain has point mutations in the MHC class II H2-IAb allele. The mouse strains and their MHC haplotypes and alloantigens are listed in Table 1 .

Table 1: Mouse MHC Haplotypes and Alloantigens Mouse Strains MHC Haplotypes H-2K H-2D I-A I-E C57BL/6J (WT) H2b b b b null B6.C-H2-Kbm1/ByJ (bm1) H2bm1 bm1 b b null B6(C)-H2-Ab1bm12/KhEgJ (bm12) H2bm12 b b bm12 null id="p-204"

[0204] C57BL/6J is a wild-type (WT) mouse strain with haplotype H2b ( FIG. 1A ). MHC haplotype antigens of C57BL/6J mice are H-2Db, H-2Kb, and I-Ab. B6.C-H2-Kbm1/ByJ (bm1) is an MHC class I mutant with a variant allele, H2-Kbm1 ( FIG. 1B ). The H2-Kbm1 allele differs from H2-Kb by 7 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide-binding groove in positions 152, 155 and 156 of the α2 domain. B6.C-H2-Kbm1/ByJ (bm1) bears the haplotype H-2Db, H-2Kbm1, and I-Ab. B6(C)-H2-Ab1bm12/KhEgJ (bm12) is an MHC class II mutant with a variant allele, H2-Ab1bm12 ( FIG. 1C ). The H2-Ab1bm12 allele differs from the H2-IAb allele by 3 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide-binding groove in positions 67, 70 and 71 of the β1 domain. B6(C)-H2-Ab1bm12/KhEgJ (bm12) bears the haplotype H-2Db, H-2Kb, and I-Abm12. id="p-205"

[0205] Antigen presenting cells (APCs, such as BMDCs) from a C57BL/6J WT mouse comprise wild type MHC alloantigens H2-Db, H2-Kb, and H2-IAb. When such APCs are pulsed by a Epeptide from human papilloma virus (HPV) protein E7, only MHC class I alloantigen, H2-Db presents the E7 peptide ( FIG. 2A ). APCs from a B6.C-H2-Kbm1/ByJ (bm1) mouse comprise wild type H2-Db which presents the E7 peptide, and wild type H2-IAb, and mutant H2-Kb which stimulates MHC class I allogeneic help ( FIG. 2B ). APCs from a B6(C)-H2-Ab1bm12/KhEgJ (bm12) mouse contains wild type H2-Db which presents the E7 peptide, and wild type H2-Kb, and mutant H2-IAb which stimulates MHC class II allogeneic help ( FIG. 2C ). FIG. 2Dindicates the genetic loci of the murine H-2 complex, also known as the murine major histocompatibility complex (MHC). id="p-206"

[0206] Syngeneic BMDCs are the BMDCs collected from one mouse strain (e.g., C57BL/6J WT), pulsed with an antigen peptide (e.g., H-2Db-restricted E743-77 peptide), and injected back to the same mouse strain (e.g., C57BL/6J WT). Semi-allogeneic BMDCs can be the BMDCs collected from one mouse strain (e.g., C57BL/6J WT), pulsed with an antigenic peptide, and injected to a different mouse strain which has at least one different MHC alloantigen (e.g., B6.C-H2-Kbm1/ByJ (bm1) or B6(C)-H2-Ab1bm12/KhEgJ (bm12)). In some embodiments, Semi-allogeneic BMDCs can be the BMDCs collected from B6.C-H2-Kbm1/ByJ (bm1) or B6(C)-H2-Ab1bm12/KhEgJ (bm12) mice, pulsed with an antigenic peptide (e.g., H-2Db-restricted E743-peptide), and injected into C57BL/6J WT mice.

Example 2: Generation of Bone-Marrow Derived Dendritic Cells (BMDCs) id="p-207"

[0207] This example illustrates a method to generate dendritic cells (DCs) from murine bone marrow (BM) progenitors. id="p-208"

[0208] Dendritic cells (DCs) were generated from bone marrow (BM) progenitors using the protocols of Lutz et al., J. Immunol. Meth. 223: 77-92 (1999) with minor modifications. Briefly, total BM cells were obtained from femurs and tibiae of 8- to 20-week-old mice (C57BL/6J, B6.C-H2-Kbm1/ByJ (bm1), or B6(C)-H2-Ab1bm12/KhEgJ (bm12)) after flushing with cell culture medium which contains RPMI-1640 (Gibco, USA) supplanted with 10 % heat-inactivated fetal bovine serum (FBS) (Sigma Aldrich, USA), 100 U/ml Penicillin-Streptomycin (Gibco, USA), 2 mM L-Glutamine (Gibco, USA), 10 mM HEPES (Gibco, USA), 1% Non-essential amino acid (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA) and 50 μM 2-mercaptoethanol (Gibco, USA) using a 3 ml syringe with a 25G needle. The BM cells were centrifuged at 1500 rpm for 5 min and then lysed red blood cells in Ammonium-Chloride-Potassium lysing buffer (Lonza, USA). The cells were strained through a 40 μm filter and washed twice with cell culture medium by centrifuging. At Day 0, 2 x 10 cells were seeded in 100 mm-bacteriological petri dishes in a total volume of ml culture medium containing 20 ng/ml recombinant murine granulocyte- macrophage colony-stimulating factor (GM-CSF; PeproTeck/Tebu, Frankfurt, Germany). Additional 10 ml of culture medium containing 20 ng/ml GM-CSF were added on Day 3. On Day 6, 8, and 10, half of the supernatant containing cells were aspirated, washed, resuspended in 10 ml fresh culture medium containing 20 ng/ml GM-CSF, and returned to the plate. A fraction of each floating and loosely adherent cell sample on Day 7, 8, 9, 10 and 12 was collected and incubated with or without 1ng/ml lipopolysaccharide (LPS, Sigma-aldrich, Heidelberg, Germany) overnight to check their proliferation and maturation. All the samples (both mature and immature cells) were measured for the expression of MHC class II (IAIE), CD11c, CD40, CD80 and CD86. The flow cytometry results showed CD11c+ IAIE+ cells were over 80% after 8 days culturing and reached over 90% after 10 days culturing with GM-CSF ( FIG. 3A ) and higher expression levels of MHC class II (IAIE), CD40, CD80, and CD86 in mature BMDCs ( FIG. 3B-E ), indicating that the majority of mature BMDCs could be used as DC vaccine in vivo.

Example 3: Generation of E7-Pulsed Mature BMDCs id="p-209"

[0209] This example illustrates a method of stimulating BMDCs using an antigen peptide and maturing BMDCs. id="p-210"

[0210] After 7-10 days of culture in medium containing 20ng/ml GM-CSF, BMDCs from C57BL/6J (WT), B6.C-H2-Kbm1/ByJ (bm1), or B6(C)-H2-Ab1bm12/KhEgJ (bm12) were collected, resuspended at 1-2 x 10 cells/ml in fresh culture medium and were incubated for 1-2 hours with a peptide from the E7 protein, E743-77 (10 µg/ml, United biosystems #241093) for antigen pulsing. The amino acid sequence of E743-77 is GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 1). The E7 peptide binds to the H-2Db MHC molecules on the BMDCs ( FIG. 2 ). These antigen-pulsed BMDCs can function as antigen presenting cells to present antigen through the H-2Db-E7 peptide complex to CD8+ cells, thus stimulating CD8+ cells to attack E7-expressing TC-1 cancer cells, eventually slowing or stopping the growth of the cancer. id="p-211"

[0211] After pulsing, BMDCs from C57BL/6J (WT), B6.C-H2-Kbm1/ByJ (bm1), or B6(C)-H2-Ab1bm12/KhEgJ (bm12) were matured with 100 ng/ml LPS overnight. After overnight incubation, the cells were detached from petri dishes by 2 mM EDTA and washed with PBS to remove the peptide and LPS and resuspended in PBS for further use. The expression of CD11c in the BMDCs were examined by flow cytometry. Over 90% of BMDCs from all three mouse strains (WT, bmand bm12) were CD11c+ after 10 days of culture with GM-CSF ( FIG. 4A ), indicating the majority of BMDCs were converted to bone fide DCs. The expression of H2-Db (B) and MHC class II (IA/IE), CD40, CD80 and CD86 (C) were also examined by flow cytometry ( FIG. 4B and 4C ), to confirm the maturation of BMDCs from all three mouse strains (WT, bm1 and bm12).

Example 4: Murine Tumor Models id="p-212"

[0212] This example illustrates that a peptide-pulsed MHC class II mutant dendritic cell vaccine has superior efficacy in a murine tumor model. id="p-213"

[0213] Autologous dendritic cell (DC) vaccines with tumor antigens have been used clinically with limited therapeutic effects. Semi-allogeneic DC-based immunotherapy is still controversial, but it can be an alternative source and more attractive than autologous DC vaccines because the "off-the-shelf" DCs can be used for multiple patients without lengthy individual manufacturing time and may provide additional "allogeneic help". Two mouse studies were performed to compare efficacy of syngeneic DC vaccines and semi-allogeneic DC vaccines and determine whether a therapeutic semi-allogeneic DC vaccine is more efficacious in tumor suppression. Female C57BL/6 mice were inoculated subcutaneously with human papillomavirus E6 and E7-expressing TC-1 cells. Syngeneic bone marrow dendritic cells (BMDCs) were generated from C57BL/6 and semi-allogeneic BMDCs were generated from two mouse strains, B6.C-H2-Kbm1/ByJ and B6(C)-H2-Ab1bm12/KhEgJ which had limited point mutations in the MHC class I H2-Kb allele or MHC class II H2-IAb allele, respectively. Each BMDC was pulsed with H-2Db-restricted E743-77 peptide and matured before injection. The mice received intradermal injections of syngeneic or one of the semi-allogeneic E7-pulsed BMDC vaccines starting 8-9 days after the TC-1 implantation. Compared with saline control, the MHC class I mutant BMDC vaccine had efficacy similar to the syngeneic BMDC vaccine in suppressing TC-1 tumor growth. However, the MHC class II mutant BMDC vaccine had efficacy significantly superior to that of the other BMDC vaccines. Thus, MHC class II semi-allogeneic BMDCs may be more effective than syngeneic DC-based cancer vaccines, presumably because the class II alloantigens induce additional T cell help for anti-tumor immunity. id="p-214"

[0214] In the first mouse tumor model study, female C57BL/6J mice (13 weeks old) were inoculated subcutaneously with TC-1 cells and later vaccinated intradermally with either syngeneic or semi-allogeneic E7-pulsed BMDC vaccines 5 times. TC-1 cells are a mouse lung cancer cell line expressing human papilloma virus (HPV) proteins E6 and E7. Eight days after the TC-1 cell inoculation, all tumor growth on mice reached around 5 mm in diameter. On Day 8, 13, 19, 23 and 28 after the TC-1 cell inoculation, mice were vaccinated intradermally 2 x 10 (1.6 x on Day 19) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ (E7-mBMDC bm1), or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). As shown in FIG. 5A , compared to the PBS control group, vaccination with the E7-pulsed BMDC WT slowed tumor growth. The E7-pulsed BMDCs of bm1 (MHC class I mutant) showed a similar effect as the E7-pulsed BMDCs (WT), but the E7-pulsed BMDCs of bm12 (MHC class II mutant) showed a greater effect than either the E7-pulsed BMDCs (WT) or the E7-pulsed BMDCs of bm1 (MHC class I mutant). A tumor on one mouse injected with the E7-pulsed BMDCs of bm12 (MHC class II mutant) had disappeared after it received the fifth vaccination ( FIG. 5B) . id="p-215"

[0215] More specific tumor measurements (tumor volume growth and tumor weight) of the mice after TC-1 cell inoculation and E7-pulsed mBMDCs vaccinations are shown in FIG. 6. Tumor volumes on Day 19, 21, 34 post-TC-1 cell inoculation indicate that all vaccinated mice exhibited slower tumor growth compared with the PBS control mice, and after the third vaccination on Day 19, the mice vaccinated with the E7-pulsed mBMDCs of bm12 (MHC class II mutant) exhibited the best response compared with all other groups ( FIGS. 6A-C ). All the mice were euthanized, and all the tumor weights were measured on Day 35. The mice vaccinated with the E7-pulsed mBMDCs of bm12 (MHC class II mutant) had significantly lighter tumors in comparison with all other three groups ( FIG. 6D ). id="p-216"

[0216] In the second mouse tumor model study, female C57BL/6J mice (9 weeks old) were inoculated subcutaneously with TC-1 cells and later vaccinated intradermally with either syngeneic or semi-allogeneic E7-pulsed BMDC vaccines for 4 times. Nine days after the inoculation, all tumor growth on mice reached around 5 mm in diameter. On Day 9, 14, 19, and after the TC-1 cell inoculation, mice were vaccinated intradermally 2 x 10 (1.6 x 10on Day 14) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from B6.C-H2-Kbm1/ByJ (E7-mBMDC bm1), or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). Similar to the first mouse tumor model study, the mice vaccinated with the E7-BMDC WT slowed tumor growth compared with the PBS control group. The mice vaccinated with the E7-BMDC bm1 (MHC class I mutant) showed a similar effect as the mice vaccinated with the E7-BMDC WT. The mice vaccinated with the E7-BMDC bm(MHC class II mutant) showed a greater effect than either the E7-BMDCs WT or the E7-BMDC bm1 (MHC class I mutant), as indicated by the tumor growth ( FIG. 7A ) of the mice. On Day after the TC-1 cell inoculation, significant differences in tumor/body weight ratio were observed between the PBS control group and the E7-pulsed BMDCs of bm12 (MHC class II mutant) group or the E7-pulsed BMDCs of bm1 (MHC class I mutant) group ( FIG. 7B ). The tumor/body weight ratio of the E7-pulsed BMDCs of bm12 (MHC class II mutant) group was also significantly lower than the E7-pulsed BMDCs (WT) group ( FIG. 7B ). The mouse body weight ( FIG. 7C ) and the tumor weight ( FIG. 7D ) measurements of the mice on Day 28 also indicate that the mice vaccinated with E7-pulsed mBMDCs of bm12 (MHC class II mutant) had a greater effect than the other three groups. id="p-217"

[0217] Conclusion: Treatment with the semi-allogeneic cancer antigen-pulsed BMDCs of bm(MHC class II mutant) was more potent than syngeneic cancer antigen-pulsed BMDCs in limiting tumor growth. This indicates that the allo-CD4+ TH response elicited by the bm12 molecule provides additional help to the BMDCs and enhances the potency of antigen presentation producing a more potent anti-cancer response.

Example 5: Murine Tumor Models with CD4 and/or CD8 T cell depletion id="p-218"

[0218] This example illustrates that CD4 T cell depletion assists semi-allogeneic cancer vaccines to limit a late-stage tumor growth in a murine tumor model. id="p-219"

[0219] Female C57BL/6J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 TC-cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter. Intraperitoneal injection of anti-CD4 antibody at an early stage of tumor growth (Early aCD4), or anti-CD8 antibody (aCD8), was started on day 6 (200 µg/mouse) after the inoculation and continued every 2-4 days (100 µg/mouse) to the end of the experiment. Intraperitoneal injection of anti-CD4 antibody at a late stage of tumor growth (late aCD4) was started on day 17 (2µg/mouse) after the inoculation and continued every 2-4 days (100 µg/mouse) to the end of the experiment. On Day 7, 12, 17, 22 and 27 after the TC-1 cell inoculation, mice were vaccinated intradermally with 2 x 10 syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT) or semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). id="p-220"

[0220] As shown in FIG. 8A , compared to the PBS control group, vaccination with the E7-BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT) slowed tumor growth, but the E7-BMDC bm12 (MHC class II mutant) with isotype control antibody (Isotype + the E7-BMDC bm12) showed a greater effect on limiting tumor growth than the E7-BMDCs WT with isotype control antibody (Isotype + the E7-BMDC WT). The mice vaccinated with E7-BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT) and the mice vaccinated with E7-BMDC WT and treated with anti-CD4 antibody at an early stage of tumor growth (Early aCD+ E7-BMDC WT) showed similar tumor growth curves ( FIG. 8B) . The mice vaccinated with E7-BMDC bm12 with isotype control antibody (Isotype + E7-mBMDC bm12) showed a great effect on limiting tumor growth ( FIG. 8C ). Administration of anti-CD4 antibody at an early stage of tumor growth to the vaccinated mice (Early aCD4 + E7-mBMDC bm12) inhibited such effect until day 17. However, further shown in FIG. 8C , administration of anti-CD4 antibody at a late stage of tumor growth to the vaccinated mice (late aCD4 + E7-mBMDC bm12) showed the best effect on suppressing tumor growth after 5th dose vaccination, indicating that CD4 depletion can contribute E7-BMDC bm12 against tumor at a late stage of tumor growth. id="p-221"

[0221] FIG. 9 shows tumor volume growth of the mice on day 14 ( FIG. 9A ), 20 ( FIG.9B ), and ( FIG. 9C ) after TC-1 cell inoculation in the CD4 or CD8 T cell depletion study. As shown in FIG. 9A , at an early stage of tumor growth, depletion of CD4 T cells significantly reduced the effect of E7-BMDC bm12 on limiting tumor growth, indicating that the allo-CD4+ Th response elicited by semi-allogeneic cancer antigen-pulsed BMDCs of bm12 (MHC class II mutant) is critical to control tumor growth at an early stage. However, at a late stage of tumor growth when CD4+ CD25+ suppressor T cells start to be upregulated in the immune system, depletion of CD4 T cells assisted E7-mBMDC bm12 vaccine to limit tumor growth, as shown in FIG. 9C . These data indicate that depletion of CD4 T cells, most likely through the depletion of CD4+ CD25+ suppressor T cells at a late stage of tumor growth, can contribute semi-allogeneic cancer vaccine in limiting tumor growth.

Example 6: Murine Tumor Models with Treg cell depletion id="p-222"

[0222] This example illustrates that Treg depletion assists semi-allogeneic cancer vaccines to limit tumor growth in a murine tumor model. id="p-223"

[0223] To further confirm whether the depletion of CD4+ CD25+ suppressor T cells or Regulatory T cells (Treg cells) could contribute E7-mBMDC bm12 to suppress tumor growth in mice, 6 groups of female B6.129(Cg)-Foxp3tm3(Hbegf/GFP)Ayr/J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 TC-1 cells. Seven days after the inoculation, all tumors on the mice reached around 5 mm long diameter. The first group of mice was injected with phosphate-buffered saline (PBS) only as a control group. Two groups of mice (2 doses of E7-mBMDC bm12) were vaccinated intradermally 2 x 10 semi-allogeneic E7-pulsed BMDCs from B6(C)-H2-Ab1bm12/KhEgJ (E7-mBMDC bm12) on day 3 and 13 after the TC-1 cell inoculation. Three groups of mice (5 doses of E7-mBMDC bm12) received the same vaccine on day 8, 13, 18, 23, and 28. These Foxp3DTR knock-in mice express the human diphtheria toxin (DT) receptor, and they are depleted for Treg cells when they are injected with DT. Among the two "2 does of E7-mBMDC bm12" groups, one group of mice (late DT + 2 doses of E7-mBMDC bm12) received late DT injection in which intraperitoneal injection of 10 µg/kg DT was started on day 15 after the inoculation, and the other group did not receive DT treatment (2 doses of E7-mBMDC bm12). Among the three "5 does of E7-mBMDC bm12" groups, one group of mice (Early DT + 5 doses of E7-mBMDC bm12) received early DT injection in which intraperitoneal injection of 10 µg/kg DT was started on day 6 after the inoculation; one group of mice (late DT + 5 doses of E7-mBMDC bm12) received late DT injection started on day 15 after the inoculation; and the last group did not receive DT treatment (5 doses of E7-mBMDC bm12). All the mice treated with DT continued receiving DT treatment every 2-4 days to the end of the experiment. id="p-224"

[0224] As shown in FIG. 10 , compared to the vaccination only with the E7-BMDC bm12 (MHC class II mutant), vaccination of the E7-BMDC bm12 combined with Treg cell depletion with early DT slowed better tumor growth suppression only at an early stage of the tumor growth. However, vaccination of either 2 doses or 5 doses of E7-BMDC bm12 combined with Treg cell depletion with late DT slowed greater suppression of tumor growth, but late Treg depletion with 5 doses of E7-mBMDC bm12 showed greater effect compare with that with 2 doses of E7-mBMDC bm12.

Compared to the mice vaccinated with E7-BMDC bm12 only (either 2 or 5 doses of E7-mBMDC bm12), the vaccinated mice treated with DT displayed a reduced tumor growth: the treatment of "early DT + 5 doses of E7-BMDC bm12" showed a slower tumor growth at an early stage, the treatment of "late DT + 2 doses E7-BMDC bm12" showed a slower tumor grow at a late stage of tumor growth, and the treatment of "late DT + 5 doses E7-BMDC bm12" showed the best effect on controlling last-stage tumor grow and also exhibited tumor suppression at the end of the experiment. These data indicate that Treg cell depletion assists semi-allogeneic cancer vaccine to limit tumor growth in mice.

Example 7: IFNγ Production in TC-1 Bearing T cells Co-cultured With BMDC Vaccines id="p-225"

[0225] This example illustrates IFNγ production in TC-1 bearing mouse T cells co-cultured with mBMDC WT, E7-mBMDCs WT, mBMDC bm12, E7-mBMDC bm12. id="p-226"

[0226] CD4 and CD8 T cells were isolated from spleens of TC-1 bearing mice 8 days after TC-inoculation and incubated at a concentration of 2 x 10 cells per well, alone or co-cultured with BMDCs (1 x 10 cells per well, with or without E7-pulsing), in a 96-well round plate for 24 hrs or hrs. The isolated CD8 T cells, either alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDCs WT (E7-WT CD8), mBMDC bm12 (bm12 CD8), or E7-pulsed mBMDC bm12 (E7-bm12 CD8), were tested for IFNγ production via intracellular staining and flow cytometry ( FIG. 11A ) and for IFNγ production in supernatant by ELISA ( FIG. 11C ). The isolated CD4 T cells, either alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDCs WT (E7-WT CD4), mBMDC bm12 (bm12 CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD4), were tested for IFNγ production via intracellular staining and flow cytometry ( FIG. 11B ) and for IFNγ production in supernatant by ELISA ( FIG. 11C ). A mix of 2 x 10 CD8 and x 10 CD4 T cells, either alone (CD8+ CD4) or co-cultured with mBMDC WT (WT CD8+CD4), E7-pulsed mBMDCs WT (E7-WT CD8+CD4), mBMDC bm12 (bm12 CD8+CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD8+CD4), were also tested ( FIGs. 11A-C ). id="p-227"

[0227] As shown in FIG. 11A,CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8) generated significantly higher level of IFNγ in comparison to a mix of CD8 and CD4 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8+CD4). CD8 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8) and a mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4) showed similar high IFNγ production to CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8). These data indicate allo-CD4+ Th response from E7-mBMDC bm12 vaccination can stimulate CD8 cells to produce high level of IFNγ. As shown in FIG. 11B,CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) generated significantly higher level of IFNγ in comparison to CD4 T cells co-cultured with E7-mBMDC WT (E7-WT CD4).

FIG. 11C showed IFNγ production in supernatant from CD4 and/or CD8 T cell culture by ELISA 72hrs after the co-culture. While no IFNγ was detected in most samples, CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) showed a good amount of IFNγ release, significantly higher than CD8 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD8). A robust IFNγ production was observed in the supernatant of the mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4), indicating co-culturing CD8 and CD4 T cells has a synergistic effect on IFNγ production.

V. References 1. Lutz MB, Kukutsch N, Ogilvie AL, Rössner S, Koch F, Romani N, Schuler G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999 Feb 1;223(1):77-92. doi: 10.1016/s0022-1759(98)00204-x. PMID: 10037236. 2. Zwaveling S, Ferreira Mota SC, Nouta J, Johnson M, Lipford GB, Offringa R, van der Burg SH, Melief CJ. Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides. J Immunol. 2002 Jul 1;169(1):350-8. doi: 10.4049/jimmunol.169.1.350. PMID: 12077264. 3. Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, Wu TC. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 1996 Jan 1;56(1):21-6. PMID: 8548765. id="p-228"

[0228] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

INFORMAL SEQUENCE LISTING E743-77: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 1).

Claims (33)

1. WHAT IS CLAIMED IS:1. An engineered mammalian dendritic cell comprising one or more exogenous alleles of at least one Major Histocompatibility Complex (MHC) class II gene.

2. The engineered mammalian dendritic cell of claim 1, wherein the one or more exogenous alleles are introduced through homologous recombination or through transfection or transduction of one or more expression vectors into the cell.

3. The engineered mammalian dendritic cell of claim 1 or 2, wherein the exogenous alleles comprise an exogenous allele of a first MHC class II gene and an exogenous allele of a second MHC class II gene.

4. The engineered mammalian dendritic cell of claim 1 or 2, wherein the exogenous alleles comprise a first exogenous allele of an MHC class II gene and a second exogenous allele of the same MHC class II gene.

5. The engineered mammalian dendritic cell of any one of claims 1 to 4, wherein the engineered mammalian dendritic cell is an engineered human dendritic cell.

6. The engineered mammalian dendritic cell of any one of claims 1 to 5, wherein the MHC class II gene comprises an HLA class II alpha subunit gene, an HLA class II beta subunit gene, or a combination thereof.

7. The engineered mammalian dendritic cell of any one of claims 1 to 6, wherein the MHC class II gene comprises an HLA-DR gene, an HLA-DP gene, an HLA-DQ gene, an HLA-DM gene, an HLA-DO gene, or a combination thereof.

8. The engineered mammalian dendritic cell of claim 7, wherein the HLA-DR gene comprises an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRBgene, an HLA-DRB5 gene, or a combination thereof.

9. The engineered mammalian dendritic cell of claim 7 or 8, wherein the HLA-DP gene comprises an HLA-DPA1 gene, an HLA-DPB1 gene, or a combination thereof.

10. The engineered mammalian dendritic cell of any one of claims 7 to 9, wherein the HLA-DQ gene comprises an HLA-DQA1 gene, an HLA-DQB1 gene, or a combination thereof.

11. The engineered mammalian dendritic cell of any one of claims 1 to 10, wherein the engineered mammalian dendritic cell comprises an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.

12. The engineered mammalian dendritic cell of any one of claims 1 to 11, wherein the cell is engineered from a cell line.

13. The engineered mammalian dendritic cell of claim 12, wherein the cell line is HL-60, THP-1, K562, MUTZ3, or an immortalized dendritic cell.

14. The engineered mammalian dendritic cell of claim 13, wherein the immortalized dendritic cell expresses HTLV-1 transactivator (Tax) protein, SV40 proteins, and/or hTERT.

15. The engineered mammalian dendritic cell of any one of claims 1 to 11, wherein the cell is engineered from a primary cell.

16. The engineered mammalian dendritic cell of claim 15, wherein the primary cell is from a patient.

17. The engineered mammalian dendritic cell of claim 16, wherein the patient has a cancer.

18. A composition comprising an engineered mammalian dendritic cell of any one of claims 1 to 17.

19. A pharmaceutical composition comprising the composition of claim 18 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, further comprising a cryoprotectant.

21. A method for semi-allogeneic dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 19 or 20.

22. The method of claim 21, wherein prior to the administering, the method further comprises: (i) obtaining an MHC class II allele profile by genotyping a plurality of MHC class II genes in a biological sample from the subject; and (ii) selecting an engineered mammalian dendritic cell for administering to the subject, wherein the engineered mammalian dendritic cell comprises one or more mismatches to the MHC class II allele profile of the subject.

23. The method of claim 21 or 22, wherein the method further comprises administering to the subject a regulatory T cell inhibitory agent (Treg agent).

24. The method of claim 23, wherein the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof.

25. The method of claim 23 or 24, wherein the Treg agent is administered after administering the pharmaceutical composition.

26. The method of any one of claims 21 to 25, wherein the subject is a human.

27. The method of claim 26, wherein the human has a cancer, wherein the engineered dendritic cell comprises a tumor-specific antigen or a fragment thereof.

28. A method for autologous dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 19 or 20, wherein the engineered mammalian dendritic cell is from a primary immune cell of the subject.

29. The method of claim 28, wherein prior to the administering, the method further comprises: (i) obtaining the primary immune cell or a plurality thereof from the subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cell to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cell into an engineered mammalian dendritic cell by: (a) introducing into the primary immune cell one or more exogenous MHC class II alleles comprising at least one mismatch to the endogenous MHC class II allele profile of the subject; and (b) introducing into the primary immune cell an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.

30. The method of claim 29, wherein (iii) further comprises: (c) incubating the primary immune cell with Lipopolysaccharide (LPS), interferon-gamma (IFN-γ), or a combination thereof.

31. The method of any one of claims 28 to 30, wherein the method further comprises administering to the subject a Treg agent.

32. The method of claim 31, wherein the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof.

33. The method of claim 31 or 32, wherein the Treg agent is administered after administering the pharmaceutical composition. For the Applicant WOLFF, BREGMAN AND GOLLER By: