Methods for enhancing the immunogenicity of cell vaccines

Abstract

This disclosure provides engineered mammalian dendritic cells expressing exogenous alleles of major histocompatibility complex (MHC) class II. Methods for using these engineered mammalian dendritic cells for immunotherapy using hemoallogeneic and autologous dendritic cells in a subject are also provided. TIFF2026518831000005.tif133160

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 456,176, filed on 31 March 2023, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] background Autologous dendritic cell (DC) vaccines containing tumor antigens are used clinically, but their therapeutic effects are limited. Immunotherapy using semi-allogeneic DCs, while still controversial, could be an alternative source and may be more attractive than autologous DC vaccines. This is because "off-the-shelf" DCs can be used for a large number of patients without requiring very long individual manufacturing times and can provide additional "allogeneic adjuncts." This disclosure addresses these needs and also provides related advantages. [Overview of the project]

[0003] overview In one aspect, the present disclosure provides engineered mammalian dendritic cells comprising one or more exogenous alleles for at least one major histocompatibility complex (MHC) class II gene.

[0004] In some embodiments, one or more exogenous alleles are introduced by homologous recombination or by transfection or transduction of one or more expression vectors into cells.

[0005] In some examples, the exogenous alleles include an exogenous allele for a first MHC class II gene and an exogenous allele for a second MHC class II gene. In other examples, the exogenous alleles include a first exogenous allele for the same MHC class II gene and a second exogenous allele for the same MHC class II gene.

[0006] In some embodiments, the engineered mammalian dendritic cells are engineered human dendritic cells. In some embodiments, the MHC class II genes include HLA class IIα subunit genes, HLA class IIβ subunit genes, or combinations thereof. In some embodiments, the MHC class II genes include HLA-DR genes, HLA-DP genes, HLA-DQ genes, HLA-DM genes, HLA-DO genes, or combinations thereof. In some embodiments, the HLA-DR genes include HLA-DRA genes, HLA-DRB1 genes, HLA-DRB3 genes, HLA-DRB4 genes, HLA-DRB5 genes, or combinations thereof. In some embodiments, the HLA-DP genes include HLA-DPA1 genes, HLA-DPB1 genes, or combinations thereof. In some embodiments, the HLA-DQ genes include HLA-DQA1 genes, HLA-DQB1 genes, or combinations thereof.

[0007] In some embodiments, the manipulated mammalian dendritic cells contain pathogen antigens, tumor-associated antigens, neoantigens, allergens, antigens targeted by autoimmune responses, or fragments thereof.

[0008] In some cases, the cells are artificially generated from a cell line. In some embodiments, the cell line is HL-60, THP-1, K562, MUTZ3, or immortalized dendritic cells. In some embodiments, the immortalized dendritic cells express HTLV-1 transactivator (Tax) protein, SV40 protein, and / or hTERT.

[0009] In other examples, cells are artificially created from primary cells. In some embodiments, the primary cells are derived from a patient. In some embodiments, the patient has cancer.

[0010] In another aspect, the Disclosure provides a composition comprising engineered mammalian dendritic cells comprising one or more exogenous alleles for at least one major histocompatibility complex (MHC) class II gene. In some embodiments, the Disclosure provides a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant.

[0011] In a further context, the Disclosure provides a method for immunotherapy using hemi-allogeneic dendritic cells in a subject, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition described herein to the subject. In some embodiments, prior to the administration step, 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 derived from the subject, and (ii) selecting engineered mammalian dendritic cells for administration to the subject, wherein the engineered mammalian dendritic cells contain one or more mismatches with the subject's MHC class II allele profile. In some embodiments, the method further comprises the step of administering a regulatory T cell inhibitor (Treg activator) to the subject. In some embodiments, the Treg activator is selected from the group consisting of antibodies, small molecules, antibody-drug conjugates, immunotoxins, peptide-drug conjugates, peptides, small interfering RNA (siRNA), siRNA conjugates, chemotherapeutic agents, and any derivatives, fragments, or fusions thereof. In some embodiments, the Treg activator is administered after the administration of the pharmaceutical composition. In some embodiments, the subject is human. In some embodiments, the human has cancer, and the manipulated dendritic cells contain tumor-specific antigens or fragments thereof.

[0012] In another aspect, the present disclosure provides a method for immunotherapy using autologous dendritic cells in a subject, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition described herein to a subject, wherein the engineered mammalian dendritic cells are derived from the subject's primary immune cells. In some embodiments, prior to the administration step, the method further comprises (i) obtaining primary immune cells or a plurality of primary immune cells from a subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cells to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cells into engineered mammalian dendritic cells by introducing one or more exogenous MHC class II alleles into the primary immune cells, which include at least one mismatch with the subject's endogenous MHC class II allele profile; and (b) introducing pathogen antigens, tumor-associated antigens, neoantigens, allergens, antigens targeted by autoimmune responses, or fragments thereof into the primary immune cells. In some embodiments, (iii) further comprises (c) incubating primary immune cells with lipopolysaccharide (LPS), interferon-γ (IFN-γ), or a combination thereof. In some embodiments, the method further comprises the step of administering a Treg activator to a target. In some embodiments, the Treg activator is selected from the group consisting of antibodies, small molecules, antibody-drug conjugates, immunotoxins, peptide-drug conjugates, peptides, small interfering RNA (siRNA), siRNA conjugates, chemotherapeutic agents, and any derivatives, fragments, or fusions thereof. In some embodiments, the Treg activator is administered after administration of a pharmaceutical composition. [Brief explanation of the drawing]

[0013] [Figure 1]The three mouse strains used in this study are shown below. (A) The C57BL / 6J mouse is wild-type (WT) with MHC haplotype H2b. (B) The B6.C-H2-Kbm1 / ByJ(bm1) mouse is an MHC class I mutant with haplotype H2bm1. The B6.C-H2-Kbm1 / ByJ mouse differs from the C57BL / 6J mouse in the Kb allele by seven nucleotides, resulting in three amino acid substitutions at positions 152, 155, and 156 along the edge of the peptide bond groove of the α2 domain. (C) The B6(C)-H2-Ab1bm12 / KhEgJ(bm12) mouse is an MHC class II mutant with haplotype H2bm12. The B6(C)-H2-Ab1bm12 / KhEgJ mouse differs from the C57BL / 6J IAB allele by three nucleotides, resulting in three amino acid substitutions at positions 67, 70, and 71 along the edge of the peptide bond groove in the β1 domain. [Figure 2A]Figure 2 shows a schematic diagram of antigen-presenting cells (APCs) containing the MHC alloantigens H2-Db, H2-Kb, and H2-IAb, as well as the gene loci of the mouse H-2 complex. The E7 peptide is a peptide derived from the human papillomavirus (HPV) protein E7 and binds only to H2Db of MHC class I. (A) APC WT contains the wild-type MHC alloantigens H2-Db, H2-Kb, and H2-IAb, with H2-Db presenting the E7 peptide. (B) APC mutant bm1 contains wild-type H2-Db and wild-type H2-IAb presenting the E7 peptide, as well as mutant H2-Kbm1 which promotes allogeneic support of MHC class I. (C) APC mutant bm12 contains wild-type H2-Db and wild-type H2-Kb presenting the E7 peptide, as well as mutant H2-IAbm12 which promotes allogeneic support of MHC class II. (D) shows the locus of the mouse H-2 complex, also known as the mouse major histocompatibility complex (MHC). Classical MHC class I contains the H-2D, H-2K, and H-2L subclasses in the K and D regions, while non-classical MHC class Ib contains the H-2Q, H-2M, and H-2T subclasses in the Q / T / M region. Classical MHC class II contains H-2A (IA) and H-2E (IE), while non-classical MHC-IIb contains the H-2P (P), H-2M (DM), and H-2O (DO) subclasses. All subclasses of MHC class II are located in the I region. MHC class III is located in the S region. [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 3A]Figure 3 shows the expression of MHC class II (IAIE), CD11c, CD40, CD80, and CD86 in immature and mature BMDCs as measured by flow cytometry. Dendritic cells (DCs) were generated from bone marrow (BM) progenitor cells of WT C57BL / 6 mice and cultured with 20 ng / ml recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7, 8, 9, 10, and 12 days prior to collection. Cells were incubated overnight in or without 100 ng / ml lipopolysaccharide (LPS, Sigma-aldrich, Heidelberg, Germany), and both mature and immature BMDCs were measured by flow cytometry. (A) shows that CD11c+IAIE+ cells exceeded 80% after 8 days of culture and exceeded 90% after 10 days of culture with GM-CSF. (B~E) shows that BMDC maturation not only promotes CD86 expression but also increases the expression of MHC class II (IAIE), CD40, and CD80. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 3D] See the explanation in Figure 3A. [Figure 3E] See the explanation in Figure 3A. [Figure 4A]Figure 4 shows the maturation of BMDCs. BMDCs derived from C57BL / 6J (WT), B6.C-H2-Kbm1 / ByJ (bm1), or B6(C)-H2-Ab1bm12 / KhEgJ (bm12) mice, cultured with GM-CSF for 10 days, were pulsed with E743-77 (10 μg / ml) for 1-2 hours, and matured overnight using LPS (100 ng / ml). The expression of CD11c, H2-Db, CD40, CD80, and CD86 in the BMDCs was examined by flow cytometry. (A) More than 90% of the BMDCs derived from all three mouse strains (WT, bm1, and bm12) were CD11c+, indicating that the majority of BMDCs were mature. The expression of H2-Db(B) as well as CD40, CD80, and CD86(C) was also investigated by flow cytometry, and similarly, mature phenotypes were observed. [Figure 4B] See the explanation in Figure 4A. [Figure 5A]Figure 5 shows the tumor volume increase curve in mice in the first mouse study. Female C57BL / 6J mice (13 weeks old) were subcutaneously inoculated with 1 × 10⁵ TC-1 cells. Eight days after inoculation, all mouse tumors reached a diameter of approximately 5 mm. On days 8, 13, 19, 23, and 28 after TC-1 cell inoculation, mice were intradermally vaccinated with 2 × 10⁶ cells (1.6 × 10⁶ cells on day 19) of either C57BL / 6J-derived syngeneic E7 pulsed BMDC (E7-mBMDC WT), B6.C-H2-Kbm1 / ByJ-derived semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm1), or B6(C)-H2-Ab1bm12 / KhEgJ-derived semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm12). The control group was injected with phosphate-buffered saline (PBS). (A) shows the tumor volume increase curves for the four groups of mice. Data are shown as mean ± SEM (n=5 or 6, as shown in the legend). Compared to the PBS control group, vaccination with E7-BMDC WT slowed tumor growth. E7-BMDC bm1 (MHC class I mutant) showed a similar effect to E7-BMDC WT. E7-BMDC bm12 (MHC class II mutant) showed a greater tumor growth limiting effect than either E7-BMDC WT or E7-BMDC bm1 (MHC class I mutant). [Figure 5B]Figure 5 shows the tumor volume increase curve of mice in the first mouse study. Female C57BL / 6J mice (13 weeks old) were subcutaneously inoculated with 1×105 TC-1 cells. On the 8th day after inoculation, the tumors of all mice reached a diameter of approximately 5 mm. On the 8th, 13th, 19th, 23rd, and 28th days after TC-1 cell inoculation, syngeneic E7-pulsed BMDCs derived from C57BL / 6J (E7-mBMDC WT), or allogeneic E7-pulsed BMDCs derived from B6.C-H2-Kbm1 / ByJ (E7-mBMDC bm1), or allogeneic E7-pulsed BMDCs derived from B6(C)-H2-Ab1bm12 / KhEgJ (E7-mBMDC bm12) were vaccinated intradermally into mice at 2×106 cells (1.6×106 cells on the 19th day). The control group was injected with phosphate-buffered saline (PBS). (B) shows the tumor volume increase curve of individual mice. In one mouse, the tumor disappeared after the fifth vaccination with E7-BMDC bm12 (MHC class II variant). [Figure 6A] Figure 6 shows the tumor volume increase of mice on various days in the first mouse study. (A) shows the tumor volume increase on the 19th day. (B) shows the tumor volume increase on the 21st day. (C) shows the tumor volume increase on the 34th day. (D) shows the tumor weight on the 35th day. PBS indicates the control group consisting of mice injected with PBS, E7-mBMDC WT indicates mice vaccinated with syngeneic E7-pulsed BMDCs derived from C57BL / 6J, E7-mBMDC bm1 indicates mice vaccinated with allogeneic E7-pulsed BMDCs derived from B6.C-H2-Kbm1 / ByJ, and E7-mBMDC bm12 indicates mice vaccinated with allogeneic E7-pulsed BMDCs derived from B6(C)-H2-Ab1bm12 / KhEgJ. The statistical significance between groups for tumor volume (A - C) or tumor weight (D) was analyzed by the Mann-Whitney test and shown as *<P = 0.05, **<P = 0.01. [Figure 6B] Refer to the description of Figure 6A. [Figure 6C] Refer to the description of Figure 6A. [Figure 6D] Refer to the description of Figure 6A. [Figure 7A] Figure 7 shows the tumor volume increase in the second mouse study. Female C57BL / 6J mice (9 weeks old) were subcutaneously inoculated with 1×105 TC-1 cells. On the 8th day after inoculation, the tumors of all mice reached a diameter of approximately 5 mm. On the 9th, 14th, 19th, and 24th days after TC-1 cell inoculation, syngeneic E7-pulsed BMDCs derived from C57BL / 6J (E7-mBMDC WT), or allogeneic E7-pulsed BMDCs derived from B6.C-H2-Kbm1 / ByJ (E7-mBMDC bm1), or allogeneic E7-pulsed BMDCs derived from B6(C)-H2-Ab1bm12 / KhEgJ (E7-mBMDC bm12) were vaccinated intradermally into the mice at 2×106 cells (1.6×106 cells on the 14th day). The control group was injected with phosphate-buffered saline (PBS). (A) shows the tumor volume increase curves of the four groups of mice. The data are shown as mean ± SEM (n = 5 or 6 as shown in the legend). Compared with the PBS control group, vaccination with E7-BMDC WT slowed tumor growth. E7-BMDC bm1 (MHC class I variant) showed an effect similar to that of E7-BMDC WT. E7-BMDC bm12 (MHC class II variant) showed a greater effect than either E7-BMDC WT or E7-BMDC bm1 (MHC class I variant). (B) shows the ratio of tumor to body weight of the mice on the 28th day after TC-1 cell inoculation. (C) shows the body weight of the mice on the 28th day after TC-1 cell inoculation. (D) shows the tumor weight of the mice on the 28th day after TC-1 cell inoculation. PBS indicates the control group consisting of mice injected with PBS, E7-mBMDC WT indicates the mice vaccinated with syngeneic E7-pulsed BMDCs derived from C57BL / 6J, E7-mBMDC bm1 indicates the mice vaccinated with allogeneic E7-pulsed BMDCs derived from B6.C-H2-Kbm1 / ByJ, and E7-mBMDC bm12 indicates the mice vaccinated with allogeneic E7-pulsed BMDCs derived from B6(C)-H2-Ab1bm12 / KhEgJ. The statistical significance between groups was analyzed by the Mann-Whitney test and shown as *<P = 0.05, **<P = 0.01. [Figure 7B] Refer to the description of Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8A] Figure 8 shows the tumor volume increase curves in mice during CD4 T cell or CD8 T cell removal studies. Female C57BL / 6J mice (10 weeks old) were subcutaneously inoculated with 1 × 10⁵ TC-1 cells. Eight days after inoculation, all mouse tumors reached a diameter of approximately 5 mm. Intraperitoneal injection of anti-CD4 antibody (early aCD4) or anti-CD8 antibody (aCD8) was started on day 6 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. Intraperitoneal injection of anti-CD4 antibody (late aCD4) was started on day 17 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. On days 7, 12, 17, 22, and 27 after TC-1 cell inoculation, mice were intradermally vaccinated with 2 × 10⁶ cells of either syngeneic E7 pulsed BMDC (E7-mBMDC WT) derived from C57BL / 6J or semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm12) derived from B6(C)-H2-Ab1bm12 / KhEgJ. The control group was injected with phosphate-buffered saline (PBS). (A) shows the tumor volume increase curves for the three groups of mice using isotype controls (isotypes). Data are shown as mean ± SEM (n=4 or 5). Compared to the PBS control group, vaccination with E7-BMDC WT and isotype control antibody (isotype + E7-BMDC WT) slowed tumor growth. However, E7-BMDC bm12 (MHC class II variant) and isotype control antibody (isotype + E7-BMDC bm12) showed a greater tumor growth restriction effect than E7-BMDC WT and isotype control antibody (isotype + E7-BMDC WT). [Figure 8B]Figure 8 shows the tumor volume increase curves in mice during CD4 T cell or CD8 T cell removal studies. Female C57BL / 6J mice (10 weeks old) were subcutaneously inoculated with 1 × 10⁵ TC-1 cells. Eight days after inoculation, all mouse tumors reached a diameter of approximately 5 mm. Intraperitoneal injection of anti-CD4 antibody (early aCD4) or anti-CD8 antibody (aCD8) was started on day 6 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. Intraperitoneal injection of anti-CD4 antibody (late aCD4) was started on day 17 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. On days 7, 12, 17, 22, and 27 after TC-1 cell inoculation, mice were intradermally vaccinated with 2 × 10⁶ cells of either syngeneic E7 pulsed BMDC (E7-mBMDC WT) derived from C57BL / 6J or semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm12) derived from B6(C)-H2-Ab1bm12 / KhEgJ. The control group was injected with phosphate-buffered saline (PBS). (B) shows the tumor volume increase curves for four groups of mice that were either injected with phosphate-buffered saline (PBS) or vaccinated with E7-BMDC WT and an isotype control antibody (isotype + E7-BMDC WT), E7-BMDC WT and an anti-CD4 antibody in the early stages of tumor growth (early aCD4 + E7-BMDC WT), or E7-BMDC WT and an anti-CD8 antibody (aCD8 + E7-BMDC WT). The groups vaccinated with E7-BMDC WT and isotype control antibodies, or E7-BMDC WT and anti-CD4 antibodies, showed similar growth curves. [Figure 8C]Figure 8 shows the tumor volume increase curves in mice during CD4 T cell or CD8 T cell removal studies. Female C57BL / 6J mice (10 weeks old) were subcutaneously inoculated with 1 × 10⁵ TC-1 cells. Eight days after inoculation, all mouse tumors reached a diameter of approximately 5 mm. Intraperitoneal injection of anti-CD4 antibody (early aCD4) or anti-CD8 antibody (aCD8) was started on day 6 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. Intraperitoneal injection of anti-CD4 antibody (late aCD4) was started on day 17 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. On days 7, 12, 17, 22, and 27 after TC-1 cell inoculation, mice were intradermally vaccinated with 2 × 10⁶ cells of either syngeneic E7 pulsed BMDC (E7-mBMDC WT) derived from C57BL / 6J or semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm12) derived from B6(C)-H2-Ab1bm12 / KhEgJ. The control group was injected with phosphate-buffered saline (PBS). (C) shows tumor volume increase curves for five groups of mice that were either injected with phosphate-buffered saline (PBS) or vaccinated with E7-BMDC bm12 and an isotype control antibody (isotype + E7-BMDC bm12), E7-BMDC bm12 and an anti-CD4 antibody in the early stages of tumor growth (early aCD4 + E7-BMDC bm12), E7-BMDC bm12 and an anti-CD4 antibody in the later stages of tumor growth (late aCD4 + E7-BMDC bm12), or E7-BMDC bm12 and an anti-CD8 antibody (aCD8 + E7-BMDC bm12). The group vaccinated with E7-BMDC bm12 and an isotype control antibody (isotype + E7-mBMDC bm12) showed a greater tumor growth limiting effect. When anti-CD4 antibodies were administered to vaccinated mice in the early stages of tumor growth (early aCD4+E7-mBMDC bm12), such effects were inhibited until day 17. When anti-CD4 antibodies were administered to vaccinated mice in the later stages of tumor growth (late aCD4+E7-mBMDC bm12), the best tumor growth inhibitory effect was observed after the fifth vaccination.(C) indicates that CD4 depletion in the late stage of tumor growth had a greater tumor growth restriction effect in mice vaccinated with E7-BMDC bm12. [Figure 9A] Figure 9 shows the tumor volume increase of mice on various days in the CD4 T cell depletion study or CD8 T cell depletion study shown in Figure 8. (A) shows the tumor volume increase on day 14. (B) shows the tumor volume increase on day 20. (C) shows the tumor volume increase on day 29. These data indicate that the vaccine effect in "early aCD4+E7-mBMDC bm12" mice was reduced by early CD4 depletion (Figure 9A). However, since late CD4 depletion enhanced the vaccine effect that restricted late tumor growth in "late aCD4+E7-mBMDC bm12" mice (Figure 9C), it was shown that the allo CD4+Th response occurred in the early stage of tumor growth and that CD4 depletion included Treg depletion in the early stage of tumor growth. The statistical significance between groups of tumor volume (A - C) was analyzed by the Mann-Whitney test and shown as *<P = 0.05. [Figure 9B] See the explanation of Figure 9A. [Figure 9C] See the explanation of Figure 9A. [Figure 10]Shows the increase in tumor volume of mice in Treg cell depletion studies. Six groups of female B6.129(Cg)-Foxp3tm3(Hbegf / GFP)Ayr / J mice (10 weeks old) were subcutaneously inoculated with 1×105 TC-1 cells. On the 7th day after inoculation, all the tumors of the mice reached a diameter of about 5 mm. In the first group of mice, only phosphate-buffered saline (PBS) was injected as a control group. In two groups of mice (2 times E7-mBMDC bm12), on the 8th and 13th days after TC-1 cell inoculation, 2×106 semi-allogeneic E7 pulsed BMDC (E7-mBMDC bm12) derived from B6(C)-H2-Ab1bm12 / KhEgJ were vaccinated intradermally. Three groups of mice (5 times E7-mBMDC bm12) were given the same vaccine on the 8th, 13th, 18th, 23rd, and 28th days. These Foxp3DTR knock-in mice express the human diphtheria toxin (DT) receptor, and Treg cells decrease when DT is injected. In one of the two "2 times E7-mBMDC bm12" groups of mice (late DT + 2 times E7-mBMDC bm12), late DT injection was performed starting on the 15th day after inoculation with an intraperitoneal injection of 10 μg / kg DT, and the other group was not given DT treatment (2 times E7-mBMDC bm12). In one of the three "5 times E7-mBMDC bm12" groups of mice (early DT + 5 times E7-mBMDC bm12), early DT injection was performed starting on the 6th day after inoculation with an intraperitoneal injection of 10 μg / kg DT, in one group of mice (late DT + 5 times E7-mBMDC bm, late DT injection was performed starting on the 15th day after inoculation, and the last group was not given DT treatment (5 times E7-mBMDC bm12). All mice that received DT treatment continued to receive DT treatment every 2 - 4 days until the end of the experiment. The data are shown as mean ± SEM (n = 4 or 5). The statistical significance between groups was analyzed by two-way ANOVA test with post hoc Tukey's multiple comparison test, and *<P = 0.05 was shown. [Figure 11A]Figure 11 shows IFNγ production in CD4 T cells or CD8 T cells from TC-1-hospitalizing mice co-cultured with mBMDC WT, E7-mBMDC WT, mBMDC bm12, or E7-mBMDC bm12. CD4 T cells and CD8 T cells were isolated from the spleen of TC-1-hospitalizing mice 8 days after TC-1 inoculation and incubated alone or co-cultured with BMDC (1 × 10⁵ cells per well, with or without E7 pulse treatment) in 96-well round plates at a concentration of 2 × 10⁵ cells per well for 24 or 72 hours. Isolated CD8 T cells were cultured alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDC WT (E7-WT CD8), mBMDC bm12 (bm12 CD8), or E7-pulsed mBMDC bm12 (E7-bm12 CD8). IFNγ production was examined by intracellular staining and flow cytometry (A), and supernatant IFNγ production was examined by ELISA (C). Isolated CD4 T cells were cultured alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDC WT (E7-WT CD4), mBMDC bm12 (bm12 CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD4). IFNγ production was examined by intracellular staining and flow cytometry (B), and supernatant IFNγ production was examined by ELISA (C). A mixture of 2 × 10⁵ CD8 T cells and 2 × 10⁵ CD4 T cells was investigated in the same manner (A-C) as a single cell (CD8+CD4) or co-cultured with mBMDC WT (WT CD8+CD4), E7-pulsed mBMDC WT (E7-WT CD8+CD4), mBMDC bm12 (bm12 CD8+CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD8+CD4). (A) shows IFNγ production by CD8 T cells, as examined by intracellular staining and flow cytometry 24 hours after co-culture.CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8) produced significantly higher levels of IFNγ compared to a mixture of CD8 T cells 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 mixture of CD8 T cells and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4) showed similar high levels of IFNγ production compared to CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8). These data indicate that the allogeneic CD4+Th response induced by E7-mBMDC bm12 vaccination can stimulate CD8 cells to produce high levels of IFNγ. (B) shows IFNγ production by CD4 T cells examined by intracellular staining and flow cytometry at 24 hours after co-culture. CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) produced significantly higher levels of IFNγ compared to CD4 T cells co-cultured with E7-mBMDC WT (E7-WT CD4). (C) shows IFNγ production in the supernatants of cultures of CD4 T cells and / or CD8 T cells examined by ELISA at 72 hours after co-culture. IFNγ was not detected in most samples, but CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD4) showed significantly more abundant IFNγ release than CD8 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8). Robust IFNγ production was observed in the supernatants of a mixture of CD8 T cells and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4). Statistical significance between groups was analyzed by one-way ANOVA test and shown as *<P = 0.05, **<P = 0.01, ***<P = 0.005, ****<P = 0.0001. [Figure 11B] See the description of Figure 11A. [Figure 11C] See the description of Figure 11A. [Modes for carrying out the invention]

[0014] Detailed explanation I. Introduction This disclosure is based in part on our findings that engineered mammalian dendritic cells (DCs) containing a semi-allogeneic major histocompatibility complex (MHC) class II allele can induce a potent immune response in a subject. In particular, this disclosure relates to engineering mammalian DCs to express one or more exogenous MHC class II alleles. Engineered mammalian DCs can be derived from cell lines and can be used as a “ready-made” semi-allogeneic vaccine. Engineered mammalian DCs can be derived from a subject and, after modification, can be returned to the subject for the treatment of cancer and / or disease as autologous immunotherapy. Engineered mammalian DCs can be derived from one subject and can be used to treat another subject as semi-allogeneic immunotherapy.

[0015] The inventors have found that allogeneic mouse 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 allogeneic BMDCs expressing one or more exogenous MHC class I alleles. DCs are antigen-presenting cells that express both MHC class I and MHC class II molecules. MHC class I molecules activate CD8+ T cells (killer T cells) to kill target cells, while MHC class II molecules activate CD4+ T cells (helper T cells) to assist the activity of other immune cells. Without being bound by any theory, the expression of at least one exogenous allele of MHC class II molecules further assists DCs by enhancing their antigen-presenting ability and inducing an allo-CD4+ Th response, thus resulting in a more potent anti-cancer response. The engineered mammalian DCs described herein can be used as allogeneic DC or autologous DC-based vaccines.

[0016] This specification describes compositions comprising engineered mammalian DCs expressing one or more exogenous MHC class II alleles, and methods for using these cells for hemoallogeneic or autologous immunotherapy targeting diseases such as cancer. Furthermore, pharmaceutical compositions and kits comprising “ready-made” engineered mammalian DCs are also provided.

[0017] II Definition Unless otherwise specifically indicated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. In addition, any method or material similar to or equivalent to those described herein may be used in the practice of this disclosure. For the purposes of this disclosure, the following terms are defined:

[0018] As used herein, the terms “a,” “an,” and “the” include not only situations involving one member but also situations involving multiple members. For example, the singular “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes multiple such cells, and a reference to “the agent” includes a reference to one or more agents known to those skilled in the art, and so on.

[0019] Generally, the terms “about” and “approximately” refer to the degree of error that is permissible for a measured quantity, taking into account the nature or precision of the measurement. Typical examples of degree of error include being within 20 percent (%), preferably 10%, and more preferably 5%, of a given value or range of values. Alternatively, particularly in biological systems, the terms “about” and “approximately” may refer to a value that is within 10 times, preferably 5 times, and more preferably 2 times, of a given value. Unless otherwise stated, the quantities given herein are approximations, meaning that the terms “about” or “approximately” may be implicitly indicated even if not explicitly stated.

[0020] The terms “subject,” “individual,” and “patient” are used synonymously herein and mean vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, rats, monkeys, humans, livestock, sports animals, and pets. Livestock include, but are not limited to, cattle, goats, pigs, sheep, dogs, horses, and rabbits. Sports animals include, but are not limited to, horses, animals of the Bovidae subfamily (calves, bulls, and castrated bulls), as well as dogs. Pet animals include, but are not limited to, dogs, cats, rabbits, rats, pigs, horses, and guinea pigs. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included.

[0021] As used herein, the term “administer” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intrafocal, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., oral cavity, sublingual, palate, gingiva, nose, vagina, rectum, or percutaneous). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous injection, and transdermal patches.

[0022] The term “treating” means an approach to obtain beneficial or desired outcomes, including, but not limited to, therapeutic benefits and / or preventive benefits. Therapeutic benefits mean any therapeutically related improvement of one or more diseases, conditions, or symptoms being treated, or any therapeutically related effect on them. Therapeutic benefits may also mean curing one or more diseases, conditions, or symptoms being treated.

[0023] The terms “effective dose” or “sufficient dose” mean the amount of manipulated mammalian cells or other composition that is sufficient to produce a beneficial or desired result. The therapeutically effective dose may vary depending on one or more of the following, which can be readily determined by those skilled in the art: the subject and disease state being treated, the subject’s weight and age, the severity of the disease state, and the mode of administration. The specific amount may vary depending on one or more of the following: the selected specific active agent, the type of target cells, the location of the target cells in the subject, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the physical delivery system in which it is carried.

[0024] For the purposes of this specification, the effective amount is determined based on such considerations that may be known in the art. This amount must be effective in achieving the desired therapeutic effect in a subject suffering from cancer or disease. The desired therapeutic effect may include, for example, improving undesirable symptoms associated with cancer or disease, preventing the onset of such symptoms before they occur, slowing the progression of symptoms associated with cancer or disease, delaying or limiting any irreversible damage caused by cancer or disease, reducing the severity of cancer or disease or curing them, or improving survival rates or accelerating recovery from cancer or disease.

[0025] The effective dose varies, in particular, depending on the type and severity of the disease being treated, as well as the treatment plan. Typically, the effective dose is determined in a well-designed clinical trial (dose-range study), and those skilled in the art are considered to know how to properly conduct such a trial to determine the effective dose. As is generally known, the effective dose varies depending on a variety of factors, including the distribution profile of the therapeutic substance (e.g., whole-cell cancer vaccine) or composition in the body, the relationship between various pharmacological parameters (e.g., half-life in the body) and undesirable side effects, as well as other factors such as age and sex.

[0026] The term “pharmaceutically acceptable carrier” means a substance that helps deliver an active agent to a cell, organism, or subject. “pharmaceutically acceptable carrier” means a carrier or excipient that can be included in the compositions of this disclosure and does not cause a significant toxicological adverse effect on the subject. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, physiological saline, Ringer's lactate solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings and colorants, liposomes, dispersions, microcapsules, cationic lipid carriers, and isotonic and absorption retardants. Carriers may also be substances that impart stability, sterility, and isotonicity to the formulation (e.g., antimicrobial preservatives, antioxidants, chelating agents, and buffers), substances that interfere with microbial activity (e.g., antimicrobial and antifungal agents such as parabens, chlorobutanol, and sorbic acid), or substances that impart food-friendly flavor to the formulation. In some cases, the carrier is an active agent that facilitates the delivery of engineered mammalian cells to target cells or tissues. Those skilled in the art will recognize that other pharmaceutical carriers may be useful in this disclosure.

[0027] As used herein, the terms “nucleic acid” or “nucleotide” mean polymers comprising at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and include DNA, RNA, and hybrids thereof. DNA may be, for example, in the form of antisense molecules, plasmid DNA, DNA-DNA double helix, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA may be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric 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 bindings, which are synthetic, natural, and unnatural and have similar binding properties to reference nucleic acids. Examples of such analogues include, non-limitingly, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids, including known analogues of natural nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise specified, individual nucleic acid sequences also implicitly encompass their conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (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)).A "nucleotide" consists of a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked via phosphate groups. "Bases" include purines and pyrimidines, which also include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogs, as well as synthetic derivatives of purines and pyrimidines. These synthetic derivatives include, but are not limited to, modifications that introduce new reactive groups, such as, non-limitingly, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0028] The term "gene" refers to a segment of DNA involved in the production of polypeptide chains. This DNA segment may include the regions before and after the coding region (leader and trailer) that are involved in the transcription / translation and regulation of the gene product, as well as intervening sequences (introns) between individual coding segments (exons).

[0029] The terms “vector” and “expression vector” refer to a nucleic acid construct, either recombinant or synthetic, that contains a set of designated nucleic acid elements that enable the transcription of a specific polynucleotide sequence in a host cell. An expression vector may be a plasmid, a viral genome, or part of a nucleic acid fragment. Typically, an expression vector contains the polynucleotide to be transcribed, functionally linked to a promoter. The term “promoter” is used herein to mean a set of nucleic acid regulatory sequences that direct the transcription of a nucleic acid. As used herein, a promoter contains the required nucleic acid sequence near the transcription start site, such as the TATA element in the case of a polymerase type II promoter. A promoter may also optionally contain distal enhancer or repressor elements, which can be located thousands of base pairs away from the transcription start site. Other elements that may be present in an expression vector include elements that enhance transcription (e.g., enhancers) and elements that terminate transcription (e.g., terminators). In this disclosure, co-expression of multiple genes (e.g., polynucleotides terminating MHC class I alleles and / or MHC class II alleles) can be achieved by simultaneous transfection of two or more vectors, the use of multiple promoters or bidirectional promoters, or the construction of bicistronic or multicistronic vectors. Gene co-expression can be promoted by using plasmids having multiple individual expression cassettes. Typically, each promoter produces a unique mRNA transcript for each gene being expressed. Bicistronic or multicistronic vectors simultaneously express two or more different proteins from the same mRNA. Bicistronic vectors may include intrasequence ribosome entry sites (IRESs) to enable translation initiation from the internal region of the mRNA. Multicistronic vectors containing one or more self-cleaving 2A peptides are advantageous because they enable gene co-expression from the same cassette.In some cases, where only a portion of a plasmid is packaged for viral delivery, or where the relative expression levels between two or more genes are important, a multicistronic vector is preferred.

[0030] "Recombinant" means 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 containing a promoter functionally linked to a second polynucleotide (e.g., a coding sequence) may contain a promoter that is heterologous to the second polynucleotide as a result of human manipulation (e.g., by the 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)). Recombinant expression cassettes (or expression vectors) typically contain polynucleotides in combinations not found in nature. For example, a human-manipulated restriction site or plasmid vector sequence may be adjacent to a promoter, or the promoter may be separated from other sequences. Recombinant proteins are expressed from recombinant polynucleotides, and recombinant cells, recombinant tissues, and recombinant organisms are those that contain recombinant sequences (polynucleotides and / or polypeptides). Recombinant cells are cells that have been modified (e.g., transfected or transformed) by recombinant nucleotides, or by expression vectors or expression cassettes.

[0031] The term "amino acid" refers to any monomeric unit that can be contained within a peptide, polypeptide, or protein. Amino acids include natural α-amino acids and their stereoisomers, as well as unnatural (non-natural) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to an isomer that has the same molecular formula and intramolecular bonds, but differs in its bonding and three-dimensional arrangement of atoms (e.g., L-amino acids and their corresponding D-amino acids).

[0032] Natural amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Natural α-amino acids include, non-limitingly, 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 combinations thereof. Stereoisomers of natural 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 combinations thereof.

[0033] Non-natural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimes, synthetic amino acids, N-substituted glycines, and N-methylamino acids, all of which function in a similar manner to natural amino acids and have either L- or D-configuration. For example, "amino acid analogs" can be non-natural amino acids such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium, which have the same basic chemical structure as natural amino acids (i.e., hydrogen, carboxyl group, and carbon bonded to the amino group) but have modified side chain groups or modified peptide skeletons. "Amino acid mimes" are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a similar manner to natural amino acids. Amino acids may be referred to by either the commonly known three-letter or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0034] In the context of describing amino acid sequences, the terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” and “homology,” as well as related terms and expressions, refer to sequences that have at least 60% sequence identity with respect to a reference sequence. Examples include sequences with 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 when compared to a reference sequence using an amino acid sequence comparison program such as BLAST with standard parameters. When performing a sequence comparison, typically one sequence acts as the reference sequence, and the test sequence is compared against it. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified if necessary, and parameters for the sequence algorithm program are specified. Default (standard) program parameters can be used, or alternative parameters can be specified. Next, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence to the reference sequence based on its program parameters. The "comparison window" includes references to a segment corresponding to one of any number of consecutive positions (20 to 600, usually about 50 to 200, more commonly about 100 to 150) within this segment, in which a sequence can be compared with a reference sequence corresponding to the same number of consecutive positions after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981; by the homology alignment algorithm of Needleman and Wunsch, 1970; by the similarity search method of Pearson and Lipman, 1988; by computer execution of these algorithms (e.g., BLAST); or by manual alignment and visual inspection.

[0035] Suitable algorithms for determining the percentage of sequence identity and sequence similarity include the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., 1990 and Altschul et al., 1977, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. The algorithm initially identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequence, match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold. These initial neighbor word hits serve as seeds to initiate a search for longer HSPs that contain them. These word hits are then extended bidirectionally along each sequence as long as the cumulative alignment score can increase. The cumulative score for nucleotide sequences is calculated using parameter M (reward score for a pair of matching residues, always greater than 0) and parameter N (penalty score for mismatched residues, always less than 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit extension in each direction is stopped if: the cumulative alignment score decreases by amount X from its maximum achieved value; the cumulative score becomes 0 or less 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 alignment. The BLASTN program (for nucleotide sequences) uses a word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and a comparison of both strands as initial settings.For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix as initial settings (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993).

[0036] The terms “polypeptide,” “peptide,” and “protein” are used synonymously herein and refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural and unnatural amino acid polymers. As used herein, these terms encompass amino acid chains of any length, including full-length proteins (i.e., alleles), in which amino acids are linked by covalent peptide bonds. As used herein, the amino acid sequence of a polypeptide is described from the N-terminus to the C-terminus. In other words, when describing the amino acid sequence of a polypeptide, the first amino acid at the N-terminus is called the “first amino acid.”

[0037] The terms “gene editing,” “genome editing,” “genomic engineering,” and “genome manipulation” are used synonymously. These terms refer to a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced within the genome of an organism. Genome editing can be site-specific. Non-limiting examples of genome editing techniques include the use of nucleases such as clustered short repeat palindromic sequences / Cas9 (CRISPR / Cas9) nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). Viral vectors such as integrase-deficient lentiviral vectors (IDLVs), adenoviruses, and adeno-associated viruses (AAVs) are typically used to deliver DNA for genome editing. Delivery techniques for genome editing are known in the art, and any approach may be used to introduce exogenous MHC alleles into the engineered mammalian cells described herein. (For example, see the overview by Yin et al., 2017. “Delivery technologies for genome editing.” Nature Review Drug Discovery. 16, 387-399).

[0038] The term “cancer” is intended to include any member of the disease class characterized by the uncontrolled proliferation of abnormal cells. This term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, as well as cancers of all stages and grades, including advanced cancer, pre-metastatic cancer, and post-metastatic cancer. Examples of various types of cancer include gynecological cancers (e.g., ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, and vulvar cancer); 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 tumor); digestive system cancers and gastrointestinal cancers, such as stomach cancer (e.g., gastric cancer), colorectal cancer, gastrointestinal stromal tumors (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, and anal cancer. This includes, but is not limited to, cancers of the hilum, bile duct, small intestine, and esophagus; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendiceal cancer; prostate cancer (e.g., prostatic adenocarcinoma); kidney cancer (e.g., renal cell carcinoma); central nervous system cancers (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancers (e.g., melanoma); osteosarcoma and soft tissue sarcoma (e.g., Ewing's sarcoma); lymphoma; choriocarcinoma; urinary tract cancers (e.g., urothelial bladder cancer); head and neck cancers; and bone marrow and hematological cancers (e.g., acute leukemia, chronic leukemia (e.g., chronic lymphocytic leukemia), lymphoma, multiple myeloma). As used herein, “tumor” includes one or more cancer cells.

[0039] The term "major histocompatibility complex" or "MHC" refers to a large locus on vertebrate DNA that contains a set of closely related polymorphic genes that encode cell surface proteins essential to the adaptive immune system.

[0040] The MHC locus is present in all jawed vertebrates and contains approximately 100 genes and pseudogenes. In humans, the MHC region is located on chromosome 6 between the adjacent gene markers MOG and COL11A2 (6p22.1–6p21.3, approximately 29Mb–33Mb on the hg38 assembly) and contains 224 genes spanning 3.6 megabase pairs (3,600,000 bases). In mice, the MHC region is located on chromosome 17.

[0041] MHC genes encode MHC molecules / proteins / antigens. The terms “MHC molecule,” “MHC protein,” and “MHC antigen” are used synonymously herein and refer to cell surface proteins encoded by MHC genes.

[0042] Human MHC is also called the human leukocyte antigen (HLA) complex (often simply HLA). Similarly, pig MHC is called porcine leukocyte antigen (SLA), bovine MHC is called bovine leukocyte antigen (BoLA), canine MHC is called canine leukocyte antigen (DLA), and so on. Mouse MHC is also called histocompatibility system 2 or simply H-2. Rat MHC is called RT1, and chicken MHC is called the B locus.

[0043] The MHC gene family is classified into three groups: MHC class I, MHC class II, and MHC class III. Only MHC class I and MHC class II genes encode MHC molecules directly involved in antigen presentation. MHC genes exhibit a high degree of polymorphism; for example, as listed in the IMGT database, HLA class I genes contain 19,031 alleles and HLA class II genes contain 7,183 alleles.

[0044] The term "allele" refers to a specific form or variant of a gene. Alleles may result from, for example, nucleotide substitutions, additions, or deletions, or they may 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's Nomenclature Committee for Factors of the HLA System. In this system, the HLA gene name is followed by a series of numerical fields. At a minimum, there are two numerical fields. As a non-restrictive example, HLA-A*02:101 represents a specific allele of the HLA-A gene. The first field, separated from the gene name by an asterisk, indicates the allele group. The second field, separated from the first field by a colon, indicates the specific HLA protein produced. In some examples, longer names are used (e.g., HLA-A*02:101:01:02N). In this example, the third numerical field indicates whether synonymous DNA substitutions are present within the coding region, and the fourth numerical field indicates differences between alleles present in the non-coding region. In several other examples, HLA allele names include a letter at the end. In the HLA allele naming convention, "N" indicates that the allele is a null allele (i.e., this allele results in a non-functional protein), "L" indicates that the allele results in lower-than-normal cell surface expression of a particular HLA protein, "S" indicates that the allele results in a soluble protein that is not present on the cell surface, "Q" indicates a questionable allele (i.e., an allele that may not affect normal expression), "C" indicates that the allele results in a protein that is present in the cytoplasm but not on the cell surface, and "A" indicates an allele that results in abnormal expression (i.e., it is uncertain whether a particular HLA protein will be expressed). Those skilled in the art will be familiar with various gene alleles and their naming conventions.

[0045] The term "allele profile" refers to a collection of alleles for one or more genes in a particular sample. The sample may be taken from a subject, a specific cell or cell type (e.g., dendritic cells), or engineered cells (e.g., dendritic cells engineered to express one or more proteins). In some examples, an allele profile may describe alleles for a single gene present in a sample (e.g., cells or cell lines obtained from a subject), or it may describe alleles present for two or more genes in the sample. As an unrestricted example, an allele profile may enumerate alleles present for an HLA class II gene in a particular sample. In diploid cells, there may be only one allele, or there may be two different alleles. In other examples, an allele profile lists alleles present for two or more genes.

[0046] The term "human leukocyte antigen (HLA)" refers to the gene complex that encodes human major histocompatibility complex (MHC) proteins, a set of cell surface proteins essential for the recognition of foreign molecules by the adaptive immune system. The HLA complex is located within the 3 Mbp range from chromosome 6p21. Human MHC class I proteins that present peptides originating from inside the cell are encoded by the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G genes. The HLA-A, HLA-B, and HLA-C genes are more pleomorphic, while the HLA-E, HLA-F, and HLA-G genes are less pleomorphic. HLA-K and HLA-L are also known to exist as pseudogenes. Furthermore, β2-microglobulin is an MHC class I protein encoded by the (B2M) gene. Non-restrictive examples of HLA-A nucleotide sequences are described in GenBank reference numbers NM_001242758 and NM_002116. Non-restrictive examples of HLA-B nucleotide sequences are described in GenBank reference number NM_005514. Non-restrictive examples of HLA-C nucleotide sequences are described in GenBank reference numbers NM_001243042 and NM_002117. Non-restrictive examples of HLA-E nucleotide sequences are described in GenBank reference number NM_005516. Non-restrictive examples of HLA-F nucleotide sequences are described in GenBank reference number NM_018950. Non-restrictive examples of HLA-G nucleotide sequences are described in GenBank reference number NM_002127. Non-restrictive examples of B2M nucleotide sequences are described in GenBank reference number NM_004048.

[0047] Human MHC class II proteins that present extracellular antigens to T lymphocytes are encoded by the HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR genes. The HLA-DM gene includes HLA-DMA and HLA-DMB. The HLA-DO gene includes HLA-DOA and HLA-DOB. The HLA-DP gene includes HLA-DPA1 and HLA-DPB1. The HLA-DQ gene includes HLA-DQA1, HLA-DQA2, HLA-DQB1, and HLA-DQB2. The HLA-DR gene includes HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. Non-exclusive examples of HLA-DMA and HLA-DMB nucleotide sequences are described in GenBank reference numbers NM_006120 and NM_002118, respectively. Non-limiting examples of nucleotide sequences for HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 are listed in GenBank reference numbers NM_01911, NM_002124, NM_022555, NM_021983, and NM_002125, respectively.

[0048] The term "semi-allogeneic" means at least one MHC class I or class II molecule expressed by the dendritic cell of interest or a dendritic cell line that is homogeneous with the recipient, and at least one MHC class I or class II molecule that is homogeneous with the recipient. "Homogeneic" refers to an MHC allele that codes for MHC molecular specificity that is consistent between the interest or dendritic cell line and the recipient, and immunologically compatible with at least one of the recipient's MHC class I or class II alleles. "Allogeneic" refers to at least one MHC class I or class II allele that codes for MHC molecular specificity that is not consistent with at least one of the recipient's MHC class I or class II alleles and is immunologically incompatible.

[0049] The term “vaccine” means a biological composition that, when administered to a subject, has the ability to induce acquired immunity in the subject against a particular pathogen or disease. Typically, one or more antigens or fragments of antigens related to the pathogen or disease of interest are administered to the subject. Vaccines may include, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins expressed from or on cells (e.g., cell surface proteins), proteins produced by organisms (e.g., toxins), or parts of organisms (e.g., viral envelope proteins). In some examples, cells are engineered to express proteins that, when administered as a vaccine, enhance the subject’s ability to acquire immunity against a particular cell type (e.g., enhance the subject’s ability to acquire immunity against cancer cells). As used herein, the terms “vaccine” or “whole-cell cancer vaccine” include, but are not limited to, the engineered mammalian cells of the Disclosure.

[0050] The term "cytokine" refers to small proteins released by cells that have a specific effect on intercellular interactions and information exchange. Generally, cytokines are known as lymphokines (e.g., cytokines produced by lymphocytes), monokines (e.g., cytokines produced by monocytes), or chemokines (e.g., cytokines produced by one leukocyte and acting on other leukocytes). Cytokines can act on cells that secrete them (e.g., autocrine action), on nearby cells (e.g., paracrine action), or on distant cells (e.g., endocrine action). In this disclosure, cytokines may include chemokines, interferons, interleukins, and / or tumor necrosis factor (TNF).For example, cytokines include early T cell activating antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36Y, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), and inter - Leukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-17 (IL-17), Interleukin-18 (IL-18), I Interleukin-23 (IL-21), Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Members of the Type I Interferon Family (IFN-α, IFNβ, IFNε, IFNκ, IFNω), Members of the Type II Interferon Family (IFNγ), Members of the Type III Interferon Family (IFNλ1 (IL-29), IFNλ2 (IL- This may include 28A), IFNλ3 (IL-28B), IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, and / or fractalkines.

[0051] The term "granulocyte-macrophage colony-stimulating factor (GM-CSF)" refers to a monomeric glycoprotein, also known as "colony-stimulating factor (CSF2)," 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 several cell types, particularly macrophages and eosinophils. As part of the immune / inflammatory cascade, GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) as well as monocytes. Monocytes then mature into macrophages and dendritic cells after tissue infiltration. A non-exclusive example of a human CSF2 nucleotide sequence (the gene encoding GM-CSF) is described in GenBank reference number NM_000758.

[0052] The term "interferon" refers to cytokines produced in response to infection or other inflammatory stimuli. Interferons are signaling proteins synthesized and released by host cells in response to pathogens (e.g., viruses, bacteria, parasites, tumor cells). Interferons are classified into three subgroups: type I interferons, type II interferons (IFNγ), and type III interferons. Functionally, these cytokines regulate the function of immune cells. Although type III interferons are structurally different from type I interferons, they share some common functions; both signal via the Janus kinase (JAK)-signaling and activator of transcription (STAT) pathway to induce transcription of interferon-activating gene (ISG) and promote the immune response. (See, for example, 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-α protein is produced by leukocytes and is primarily involved in the innate immune response. Genes encoding IFN-α protein include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.Non-limiting examples of human nucleotide sequences for IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21 are described in GeneBank 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. For example, the gene IFNA2 encodes the IFN-α2a, IFN-α2b, and IFN-α2c variants. As used herein, the terms “IFN-α” and “IFN-α2” are used synonymously and refer to the interferon proteins IFN-α2a or IFN-α2b. Type III interferon proteins include interferon λ1 (IFNλ1(IL-29)), interferon λ2 (IFNλ2(IL-28A)), interferon λ1 (IFNλ3(IL-28B)), and interferon λ4 (IFNλ4). Members of the interferon λ family transmit signals via a common IL-10 receptor subunit 2 (IL-10R2). Human interferon λ proteins are encoded by four IFNL genes, namely IFNL1(IL29), IFNL2(IL28A), IFNL3(IL28B), and IFNL4.

[0053] The term "costimulatory molecules" refers to cell surface molecules that amplify or counteract the initial activation signals delivered to T cells from the T cell receptor (TCR) after interaction with the antigen / major histocompatibility complex (MHC). Typically, costimulatory molecules can influence T cell differentiation and fate. Costimulatory molecules belong to three major families: the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family. (e.g., Rodriguez-Manzanet, Roselynn et al. “The costimulatory role of TIM molecules.” Immunological reviews vol. 229,1 (2009):) See 259-70.) Exemplary co-stimulatory molecules and ligands include, but are not limited to, CD28 and ligand B7-1 (CD80), CTLA-4, PDL-1, or B7-2 (CD86), CTLA-4 and ligand B7-1 (CD80) or B7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (also known as CD154), OX40 and ligand OX40L, GITR and ligand GITRL, TIM-1 and ligand TIM-1, TIM-4, IgA, or phosphatidylserine (PtdSer), TIM-2 and ligand H-ferritin or semaphorin 4A (Sem4A), and TIM-4 and ligand phosphatidylserine (PtdSer).In the context of this disclosure, co-stimulatory molecules may include the CD86 molecule (CD86), the CD80 molecule (CD80), the 4-1BB ligand molecule (4-1BBL, also known as CD137L), the ICOS ligand molecule (ICOS-L), the CD70 molecule (CD70, also known as CD27L), the CD40 molecule (CD40), the OX40 ligand molecule (OX40L), the GITR ligand molecule (GITRL), the TIM-4 molecule (TIM-4), the LIGHT molecule (LIGHT), the ICAM1 molecule (ICAM1), the LFA3 molecule (LFA3), the CD30 molecule (CD30), and combinations thereof (see, for example, Figure 5).

[0054] Regulatory T cells, or Tregs, or suppressor T cells, are essential for establishing and maintaining homeostasis and autoimmune tolerance. + CD25 +Treg cells represent a highly immunosuppressive subset of T cells. Tregs can inhibit T cell proliferation and cytokine production, thus preventing autoimmunity. Treg cells express the biomarkers CD4, CD25, and forkhead box protein P3 (FoxP3). CD4 is a marker for helper T cells, as well as for a certain population of Treg cells originating from the thymus. 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 suppresses the expression of several cytokines, including IL-2, IL-4, and IFNγ, while activating the IL-2 receptor (CD25), cytotoxic T lymphocyte-associated protein-4 (CTLA4), and glucocorticoid-inducible TNF receptor (GITR). FoxP3 is a master protein involved in the differentiation and function of regulatory T cells and can be used as a Treg molecular marker (see, e.g., Science (2003) 299: 1057-61). Detailed descriptions and information regarding human CD4+CD25+ regulatory T cells can be found in the following references, all of which are incorporated herein 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.

[0055] The terms “regulatory T cell inhibitor,” “Treg inhibitor,” and “Treg activator” mean an activator that (1) inhibits or reduces the activity or function of regulatory T cells; (2) reduces the population of regulatory T cells in a subject (in one embodiment, the reduction may be temporary, e.g., for hours, a day, a few days, a week, or several weeks); or (3) substantially removes or eliminates the population of regulatory T cells in a subject (in one embodiment, the removal or elimination may be temporary, e.g., for hours, a day, a few days, a week, or several weeks). Treg activators can reduce the suppression of immune system activation and reduce the prevention of autoreactivity. Exemplary Treg activators include, but are not limited to, compounds, antibodies, antibody fragments, or chemicals that target Treg cell surface markers (e.g., CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), CTLA4, CCR4, CCR8, FOXP3, LAG3, CD103, NRP-1, glucocorticoid-inducible TNF receptor (GITR), galectin-1, TNFR2, or TGF-βR1). In certain embodiments, Treg activators target Treg cell surface markers involved in Treg activation, and therefore Treg inhibitors prevent Treg activation. Exemplary Treg activators include, but are not limited to, antibodies, fusion proteins, ONTAK, HuMax-Tac, Zenapax, or MDX-010, aptamers, siRNA, ribozymes, and antisense oligonucleotides. Administration of Treg activators or derivatives can interfere with the action of their targets, such as Treg cell surface markers. Treg activators may have a toxic moiety bound to them, and as a result, once the inhibitor is internalized, the bound toxic moiety can kill regulatory T cells.

[0056] The term "tumor antigen" refers to antigenic substances produced within tumor cells that can trigger an immune response in the host. Generally, tumor antigens refer to tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). Typically, TSAs are present only in cancer cells and not in healthy (e.g., non-cancerous) cells. TSAs can arise from oncogenic driver mutations that produce novel peptide sequences (e.g., neoantigens). A non-limiting example of a TSA is alpha-fetoprotein (AFP), which is expressed in germ cell tumors and hepatocellular carcinoma. TAAs may be present at high levels in tumor cells and at low levels in healthy cells. A non-limiting example of a TAA is melanoma-associated antigen (MAGE), which is expressed in the testes along with malignant melanoma.

[0057] The term “survival” refers to a period of time after a diagnosis of a disease and / or the commencement or completion of a particular course of treatment for the disease (e.g., cancer). The term “overall survival” includes a clinical endpoint that represents a patient who has survived for a specified period of time after being diagnosed with a disease, such as cancer, or after receiving treatment for it. The term “disease-free survival” includes the length of time a patient has lived without signs of the disease (e.g., without a known recurrence) after treatment for a particular disease (e.g., cancer). In certain embodiments, disease-free survival is a clinical parameter used to assess the effectiveness of a particular treatment and is typically measured in units of one or five years. The term “progression-free survival” includes the length of time a patient has lived with the disease (e.g., cancer) during and after treatment for the disease without new symptoms of the disease appearing. In some embodiments, survival is expressed as a median or mean.

[0058] III. Detailed Description of the Embodiments In one aspect, the present disclosure provides engineered mammalian dendritic cells comprising one or more exogenous alleles for MHC class II genes. In some embodiments, the engineered mammalian dendritic cells comprise one exogenous allele for an MHC class II gene. In other embodiments, the engineered mammalian dendritic cells comprise multiple exogenous MHC class II alleles. In some examples, the engineered mammalian dendritic cells comprise a first exogenous allele for a first MHC class II gene and a second exogenous allele for a second MHC class II gene. In some examples, the engineered mammalian dendritic cells comprise a first exogenous allele for a certain MHC class II gene and a second exogenous allele for the same MHC class II gene. In some embodiments, the engineered mammalian dendritic cells further comprise one or more exogenous alleles for an MHC class I gene.

[0059] In one aspect, one or more exogenous alleles are introduced into mammalian dendritic cells by homologous recombination. In some embodiments, one or more exogenous MHC class II alleles are introduced into mammalian dendritic cells by homologous recombination. In some examples, homologous recombination replaces the endogenous allele of an MHC class II gene with an exogenous allele. In other examples, homologous recombination inserts an exogenous allele into an MHC class II gene without removing the associated endogenous allele. In other embodiments, one or more exogenous MHC class I alleles are also introduced into mammalian dendritic cells by homologous recombination.

[0060] In some embodiments, homologous recombination is induced by nucleases that cause double-strand breaks (DSBs) at specific sites within the genome. These nucleases may be endonucleases, zinc finger nucleases (ZENs), transcription activator-like effector nucleases (TALENs), site-specific recombinases, transposases, topoisomerases, and their modified derivatives and variants. A description of nucleases that may be used in this disclosure is further provided herein. In some embodiments, the nuclease may be an RNA-guided nuclease, such as a clustered short repeat palindrome (CRISPR) nuclease with regularly spaced intervals.

[0061] In another aspect, one or more exogenous alleles are introduced into cells by transfection or transduction of one or more expression vectors. In some embodiments, one or more exogenous MHC class II alleles are introduced into cells by transfection or transduction of one or more expression vectors. In some examples, one or more exogenous MHC class II alleles are introduced into cells by transfection of one or more expression vectors. In some embodiments, transfection of one or more expression vectors containing MHC class II alleles into cells includes transfection of plasmids or expression cassettes. In other examples, one or more exogenous MHC class II alleles are introduced into cells by transduction of one or more expression vectors. In some embodiments, transduction of one or more expression vectors containing MHC class II alleles into cells includes, but is not limited to, transduction of viral vectors such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors. In some embodiments, only one exogenous MHC class II allele is introduced into a cell by transfection or transduction of a single vector. In other embodiments, multiple exogenous MHC class II alleles are introduced into a cell. In some examples, all exogenous MHC class II alleles can reside on the same vector. In other examples, each exogenous MHC class II allele can reside on a separate vector. In yet another example, two, three, four, five, six, or more exogenous MHC class II alleles can reside on the same vector. Any combination of exogenous alleles in a single vector and any number of vectors in a cell are acceptable. A description of expression vectors and transfection or transduction methods that may be used in this disclosure is further provided herein.In certain embodiments, two exogenous MHC class II alleles selected from HLA-DR, HLA-DP, and / or HLA-DQ alleles are introduced into cells using the same vector or separate vectors. In other embodiments, one or more exogenous MHC class I alleles are also introduced into mammalian dendritic cells by transfection or transduction of one or more expression vectors. In certain examples, all exogenous MHC class II alleles may be present on one vector, and all exogenous MHC class I alleles may be present on another vector.

[0062] In one aspect, the engineered mammalian dendritic cells described herein are engineered human dendritic cells. In other aspects, engineered mammalian dendritic cells are derived from non-human dendritic cells. In some embodiments, non-human dendritic cells may be derived from mice, rats, monkeys, livestock, sports animals, or pet animals. Non-limiting examples of livestock include cattle, goats, pigs, sheep, dogs, horses, and rabbits. Non-limiting examples of sports animals include horses, cattle (calves, bulls, and castrated cattle), as well as dogs. Non-limiting examples of pet animals include dogs, cats, rabbits, rats, pigs, horses, and guinea pigs.

[0063] A Human MHC gene HLA class II gene In one aspect, the present disclosure provides engineered human dendritic cells comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) exogenous HLA class II alleles for one or more MHC class II genes. In some embodiments, the MHC class II gene is an HLA class IIα subunit gene. In other embodiments, the MHC class II gene is an HLA class IIβ subunit gene. In certain embodiments, the MHC class II gene is a combination of an HLA class IIα subunit gene and an HLA class IIβ subunit gene.

[0064] In other embodiments, MHC class II genes are HLA-DR genes, HLA-DP genes, HLA-DQ genes, HLA-DM genes, and / or HLA-DO genes.

[0065] In some embodiments, the HLA-DR gene is the HLA-DRA gene, HLA-DRB1 gene, HLA-DRB3 gene, HLA-DRB4 gene, and / or HLA-DRB5 gene. In certain embodiments, the engineered human dendritic cell contains one or more exogenous alleles for one or more (e.g., one, two, three, four, five, or more) HLA-DR genes. In some examples, the HLA-DP gene is the HLA-DPA1 gene. In other examples, the HLA-DP gene is the HLA-DPB1 gene. In certain examples, the engineered human dendritic cell contains one or more exogenous alleles for both the HLA-DPA1 gene allele and the HLA-DPB1 gene allele. In some examples, the HLA-DQ gene is the HLA-DQA1 gene. In other examples, the HLA-DQ gene is the HLA-DQB1 gene. In certain cases, the engineered human dendritic cells contain one or more exogenous alleles for both the HLA-DQA1 gene allele and the HLA-DQB1 gene allele. In some cases, the HLA-DM gene is the HLA-DMA gene. In other cases, the HLA-DM gene is the HLA-DMB gene. In certain cases, the engineered human dendritic cells contain one or more exogenous alleles for both the HLA-DMA gene allele and the HLA-DMB gene allele. In some cases, the HLA-DO gene is the HLA-DOA1 gene. In other cases, the HLA-DO gene is the HLA-DOB1 gene. In certain cases, the engineered human dendritic cells contain one or more exogenous alleles for both the HLA-DOA1 gene allele and the HLA-DOB1 gene allele.

[0066] 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-DRB4 alleles 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. Manipulated human dendritic cells of this disclosure may contain one or more (e.g., one, two, three, four, five, or more) exogenous alleles for HLA-DRB3 / 4 / 5 alleles.

[0067] Examples of appropriate HLA-DPA1 alleles include, but are not limited to, HLA-DPA1*01:05, HLA-DPA1*02:08, and HLA-DPA1*04:05. Examples of appropriate HLA-DPB1 alleles include, but are not limited to, HLA-DPB1*32:01 and HLA-DPB1*1454:01. Examples of appropriate HLA-DQA1 alleles include, but are not limited to, HLA-DQA1*01:06, HLA-DQA1*02:29, and HLA-DQA1*06:04. Examples of appropriate HLA-DQB1 alleles include, but are not limited to, HLA-DQB1*05:04, HLA-DQB1*06:100, and HLA-DQB1*04:95. Examples of appropriate 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 appropriate 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.

[0068] HLA class I gene In one aspect, the present disclosure provides engineered human dendritic cells further comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) exogenous HLA class I alleles for one or more MHC class I genes. 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.

[0069] Examples of appropriate 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. The engineered human dendritic cells of this disclosure may contain one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more) exogenous HLA-A alleles.

[0070] Examples of appropriate 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. The manipulated human dendritic cells of this disclosure may contain 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.

[0071] Examples of appropriate 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. The manipulated human dendritic cells of this disclosure may contain 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.

[0072] MHC gene of mouse B The mouse MHC consists of 11 subclasses. "Classical MHC class I" (also called MHC-Ia) includes subclasses H-2D, H-2K, and H-2L, located in the K and D regions of the mouse H-2 locus (Figure 2D). "Non-classical MHC class I" (MHC-Ib) includes subclasses H-2Q, H-2M, and H-2T in the Q / T / M region. "Classical MHC class II" (MHC-IIa) includes subclasses H-2A (IA) and H-2E (IE), while "Non-classical MHC class II" (MHC-IIb) includes H-2P (P), H-2M (DM), and H-2O (DO). All subclasses of mouse MHC class II are located in the I region. Mouse MHC class III is located in the S region.

[0073] Mouse MHC class I molecules consist of a 45kD highly glycosylated heavy chain non-covalently bonded to a 12kD 2-microglobulin polypeptide, which is also present in the serum in a free state. Mouse MHC class II antigens are composed of a 33kD chain and a 28kD chain.

[0074] MHC class I molecules are expressed in almost all nucleated cells. They play a crucial role in presenting altered autocellular antigens (virus-infected cells or tumor cells) to CD8+ cytotoxic T cells. MHC class II molecules are expressed on antigen-presenting cells (such as B cells, monocytes / macrophages, dendritic cells, and Langerhans cells). They are involved in presenting processed peptide antigens to CD4+ cells.

[0075] In one aspect, the present disclosure provides engineered mouse dendritic cells comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) exogenous MHC class II alleles for one MHC class II gene or multiple MHC class II genes. In another aspect, the engineered mouse dendritic cells further comprise one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) exogenous MHC class I alleles for one MHC class I gene or multiple MHC class I genes.

[0076] MHC haplotypes of mouse strains Laboratory mice are inbred, each strain is homozygous, and has a unique MHC haplotype. The MHC haplotypes of these strains are represented by lowercase letters (such as a, b, d, k, q, s, etc.). For example, the MHC haplotype antigens of BALB / c mice are H-2K d , H-2D d , H-2L d , H2-IA d , and H2-IE d . The MHC haplotype antigens of C57BL / 6J mice are H2-D b , H2-K b , and H2-IA b . Some variant mouse strains are also produced in the laboratory. For example, B6.C-H2-K bm1 / ByJ (bm1) is derived from a spontaneous variant (BALB / cBy×C57BL / 6By)F1 and is produced by introgressing the H2 bm1 allele on chromosome 17 into the genetic background of C57BL / 6By over 10 generations. The variant allele H2-K bm1 differs from H2-K b by 7 nucleotides, resulting in three amino acid substitutions along the edge of the peptide-binding groove at positions 152, 155, and 156 of the α2 domain. B6(C)-H2-Ab1 bm12 / KhEgJ(bm12) is also an H2-Ab1 derived from the naturally occurring variant (C57BL / 6Kh×BALB / cKh)F1. bm12 The allele is created by transferring the gene into a C57BL / 6Kh genetic background over 10 generations. This variant MHC class II allele is H2-Ab1 b Three nucleotides are different, resulting in three amino acid substitutions (Ile67Phe, Arg70Gln, Thr71Lys) along the edge of the peptide bond groove of the β1 domain.

[0077] C Selected MHC allele constructs In some embodiments, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) include one or more exogenous MHC class II genes selected from any MHC class II gene, its codon-optimized version, its variant, or fragments thereof. For example, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) may include recombinant polynucleotides encoding one, two, three, four, or more MHC class II genes driven by one or more promoters. In some embodiments, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) further include one or more exogenous MHC class I genes selected from any class I gene, its codon-optimized version, its variant, or fragments thereof. For example, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) may include recombinant polynucleotides encoding one, two, three, four, or more MHC class I genes driven by one or more promoters.

[0078] In some embodiments, the engineered mammalian dendritic cell (e.g., engineered human dendritic cell) comprises one exogenous MHC class II gene selected from any MHC class II gene, its codon-optimized version, its variant, or a fragment thereof. In other embodiments, the engineered mammalian dendritic cell (e.g., engineered human dendritic cell) comprises at least two exogenous MHC class II genes selected from any class II gene, its codon-optimized version, its variant, or a fragment thereof. For example, the engineered mammalian dendritic cell (e.g., 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.

[0079] In some embodiments, the engineered mammalian dendritic cell (e.g., engineered human dendritic cell) further comprises one exogenous MHC class I gene selected from any MHC class I gene, a codon-optimized version thereof, a variant thereof, or a fragment thereof. In other embodiments, the engineered mammalian dendritic cell (e.g., 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 thereof, a variant thereof, or a fragment thereof. For example, the engineered mammalian dendritic cell (e.g., engineered human dendritic cell) may comprise one recombinant polynucleotide encoding a first MHC class I gene and a second recombinant polynucleotide encoding a second MHC class I gene.

[0080] D. Cytokines and / or co-stimulatory molecules In one aspect, the present disclosure provides engineered mammalian dendritic cells (e.g., engineered human dendritic cells) further comprising one or more recombinant polynucleotides encoding cytokines, co-stimulatory molecules, variants thereof, fragments thereof, or combinations thereof.

[0081] Cytokines can be chemokines, interferons, interleukins, or tumor necrosis factors. Cytokines include early T cell activating antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36Y, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), and interleukin-4 (IL-4). Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-9 (IL-9), Interleukin-10 (IL-10), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-17 (IL-17), Interleukin-5 Ikin-18 (IL-18), Interleukin-21 (IL-21), Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Interferon-α (IFN-α), Interferon-λ1 (IFNλ1 (IL-29)), Interferon-λ2 (IFNλ2 (IL-28A)), Interferon-λ3 (IFNλ3 (IL-28B)), I The following may be selected: etherferon λ4 (IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, or fractalkine.

[0082] The co-stimulatory molecule may be selected from at least one of the following: 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, also known as 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.

[0083] In some embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules can be introduced into cells by homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules can be introduced into cells by transfection or transduction of one or more expression vectors.

[0084] In some embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules are located on one or more intracellular vectors, and one or more exogenous MHC alleles are located within the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules are located within the genome of the cell, and one or more exogenous MHC alleles are located on one or more intracellular vectors. In some embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules and one or more exogenous MHC alleles are located within the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules and one or more exogenous MHC alleles are located on one or more intracellular vectors.

[0085] In some examples, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules may reside on the same vector as one or more exogenous MHC alleles. In other examples, one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules may reside on a vector separate from one or more exogenous MHC alleles. As a non-limiting example, a cell may include (a) a vector containing one or more exogenous HLA class II alleles selected from HLA-DR, HLA-DP, and / or HLA-DQ alleles; and (b) a vector containing one or more recombinant polynucleotides encoding cytokines and / or costimulatory molecules. As another non-limiting example, a cell may further include (c) a vector containing one or more exogenous HLA class I alleles.

[0086] E xenoantigen In one aspect, the present disclosure provides engineered mammalian dendritic cells (e.g., engineered human dendritic cells) further comprising one or more recombinant polynucleotides encoding heterogeneous antigens (e.g., pathogen antigens, tumor-associated antigens, neoantigens, allergens, antigens targeted by immune responses), variants thereof, or fragments thereof.

[0087] In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and / or antigen peptide can be introduced into cells by homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and / or antigen peptide can be introduced into cells by transfection or transduction of one or more expression vectors.

[0088] In some embodiments, one or more recombinant polynucleotides encoding a heterogeneous antigen and / or antigen peptide are located on one or more intracellular vectors, and one or more exogenous MHC alleles are located within the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding a heterogeneous antigen and / or antigen peptide are located within the genome of the cell, and one or more exogenous MHC alleles are located on one or more intracellular vectors. In some embodiments, one or more recombinant polynucleotides encoding a heterogeneous antigen and / or antigen peptide, as well as one or more exogenous MHC alleles, are located within the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding a heterogeneous antigen and / or antigen peptide, as well as one or more exogenous MHC alleles, are located on one or more intracellular vectors.

[0089] In some examples, one or more recombinant polynucleotides encoding heterologous antigens and / or antigenic peptides may reside on the same vector as the exogenous MHC alleles. In other examples, one or more recombinant polynucleotides encoding heterologous antigens and / or antigenic peptides may reside on a vector separate from the exogenous MHC alleles. As a non-limiting example, a cell may include (a) a vector containing one or more HLA class II alleles selected from HLA-DR, HLA-DP, and / or HLA-DQ alleles; and (b) a vector containing recombinant polynucleotides encoding intrinsic antigenic peptides of pathogenic antigens, tumor-associated antigens, neoantigens, allergens, or antigens targeted by an immune response. As another non-limiting example, a cell may further include (c) a vector containing one or more exogenous HLA class I alleles (e.g., HLA-A alleles).

[0090] In another aspect, the present disclosure provides engineered mammalian dendritic cells (e.g., engineered human dendritic cells) further comprising one or more heterologous antigens (e.g., pathogen antigens, tumor-associated antigens, neoantigens, allergens, antigens targeted by immune responses), their variants, or fragments thereof. In some embodiments, one or more antigens and / or antigenic peptides can be introduced into cells by incubating the antigens and / or antigenic peptides together with the cells in the same culture.

[0091] In some embodiments, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) are derived from primary patient cells and pulsed with heterologous antigens and / or antigenic peptides of pathogens affecting the patient. In some embodiments, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) are derived from cancer patients and pulsed with tumor-specific antigens and / or fragments thereof. In some embodiments, engineered mammalian dendritic cells (e.g., engineered human dendritic cells) derived from primary patient cells are further incubated with lipopolysaccharide (LPS) after pulse treatment.

[0092] F dendritic cells In some embodiments, the engineered mammalian dendritic cells are derived from dendritic cell lines (e.g., human dendritic cell lines). Non-limiting examples of human dendritic cell lines include the following cell lines and their subclones: HL-60, THP-1, K562, MUTZ3, or immortalized dendritic cells. In some examples, immortalized dendritic cells express HTLV-1 transactivator (Tax) protein, SV40 protein, and / or hTERT.

[0093] In other embodiments, engineered mammalian dendritic cells are artificially created from the patient's primary cells, such as blood or biopsy samples derived from the patient. In some cases, the patient has cancer.

[0094] G Homologous recombination In one aspect, the engineered mammalian dendritic cells described herein contain one or more exogenous alleles in their genomic DNA. In some embodiments, one or more exogenous MHC class II alleles are introduced into the cell by homologous recombination. In some embodiments, homologous recombination replaces an endogenous allele of an MHC class II gene with an exogenous allele. In other embodiments, homologous recombination inserts an exogenous allele into an MHC class II gene.

[0095] As described herein, nucleases can be used to induce double-strand breaks (DSBs) at specific sites in the genome and thereby induce homologous recombination. Examples of nucleases include, but are not limited to, endonucleases, zinc finger nucleases (ZENs), transcription activator-like effector nucleases (TALENs), site-specific recombinases, transposases, topoisomerases, and their modified derivatives and variants. In some embodiments, the nuclease may be an RNA-guided nuclease, such as a short repeat palindrome (CRISPR) nuclease clustered at regular intervals.

[0096] Cas endonuclease In some embodiments, the nucleases used in the methods and compositions of this disclosure are CRISPR-related (Cas) proteins. A Cas protein refers to an RNA-guided double-strand DNA binding nuclease protein or nickase protein. Wild-type Cas nucleases have two functional domains, e.g., RuvC and HNH, which cleave different DNA strands. When both functional domains are active, the Cas protein can induce double-strand breaks in genomic DNA (target nucleic acid). Cas proteins are found in the genera Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, and Flavobacterium. The Cas protein may contain one or more catalytic domains derived from bacteria belonging to the group consisting of *Tylenolum*, *Sphaerochaeta*, *Azospirillum*, *Gluconacetobacter*, *Neisseria*, *Roseburia*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, and *Campylobacter*. In some embodiments, the Cas protein can be a fusion protein, for example, the two catalytic domains may be derived from different bacterial species.

[0097] Non-exclusive 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, and Csm5. This includes Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs, their variants, their mutants, and their derivatives. Cas proteins have three main types (Type I, Type II, and Type III) and 10 subtypes, including 5 Type I proteins, 3 Type II proteins, and 2 Type III proteins (see, for example, 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 wild-type Cas9 polypeptide of Streptococcus pyogenes is described, for example, NBCI reference sequence number NP_269215, and the amino acid sequence of the wild-type Cas9 polypeptide of Streptococcus thermophilus is described, for example, NBCI reference sequence number WP_011681470.

[0098] Cas proteins, such as Cas9 nuclease, can originate from a variety of bacterial species, including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, and Solobacterium mourei.moorei), Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum Lactobacillus bifidum), Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coriniformis subspecies torucens coryniformis subsp.Torquens), Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus 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 szygii syzygii), Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorance lavamentivorans), Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp.Multocida, Sutterella wadsworthensis, proteobacteria, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0099] In some embodiments, the Cas protein can be a high-fidelity or enhanced-specificity Cas9 polypeptide variant exhibiting reduced off-target activity and robust on-target cleavage. Non-limiting examples of Cas9 polypeptide variants with improved on-target specificity include SpCas9(K855A), SpCas9(K810A / K1003A / R1060A) (also known as eSpCas9(1.0)), and the SpCas9(K848A / K1003A / R1060A) (also known as eSpCas9(1.1)) variant described in Slaymaker et al., Science, 351(6268):84-8 (2016), as well as variants containing one, two, three, or four of the following mutations: N497A, R661A, Q695A, and Q926A, as described in Kleinstiver et al., Nature, 529(7587):490-5 This includes SpCas9 variants described in (2016) (for example, SpCas9-HF1 contains all four mutations).

[0100] Guide RNA The Cas protein can be guided to a target nucleic acid by a guide RNA (gRNA). gRNAs can be a pair of gRNAs or a single continuous sequence of two innate guide RNAs (crRNA and tracrRNA) that have been manipulated. A gRNA may contain a guide sequence that directs the Cas protein to the target nucleic acid (e.g., the crRNA equivalent of the gRNA) and a scaffold sequence that interacts with the Cas protein (e.g., the tracrRNA equivalent of the gRNA). gRNAs can be selected using software. Non-limiting examples of considerations for gRNA selection may include, for example, the PAM sequence for the Cas protein used and strategies to minimize off-target modifications. Tools such as NUPACK® and CRISPR design tools can provide sequences for preparing gRNAs, evaluating the efficiency of target modification, and / or evaluating cleavage at off-target sites.

[0101] Guide array The guide sequence in the gRNA may be complementary to a specific sequence within the target nucleic acid (e.g., one allele for an MHC class II gene). The PAM sequence may follow the 3' end of the target nucleic acid sequence. The target nucleic acid consists of approximately 20 nucleotides upstream of the PAM sequence. Generally, the Cas9 protein or its variants cleave approximately 3 nucleotides upstream of the PAM sequence. The guide sequence in the gRNA may be complementary to either strand of the target nucleic acid.

[0102] In some embodiments, the guide sequence of the gRNA contains approximately 100 nucleic acids at the 5' end of the gRNA, which can direct the Cas protein to the target nucleic acid site using RNA-DNA complementary base pairing. In some embodiments, the guide sequence contains approximately 20 nucleic acids at the 5' end of the gRNA, which can direct the Cas protein to the target nucleic acid site using RNA-DNA complementary base pairing. In other embodiments, the guide sequence contains fewer than 20 nucleic acids complementary to the target nucleic acid site, e.g., 19, 18, 17, 16, 15, or fewer. In some examples, the guide sequence in the gRNA contains at least one nucleic acid mismatch in the complementary region to the target nucleic acid site. In some examples, the guide sequence contains approximately 1 to approximately 10 nucleic acid mismatches in the complementary region to the target nucleic acid site.

[0103] Scaffold arrangement The scaffold sequence within gRNA can act as a protein-binding sequence that interacts with the Cas protein or its variants. In some embodiments, the scaffold sequence within gRNA may contain two complementary nucleotide stretches that hybridize with each other to form a double-stranded RNA double helix (dsRNA double helix). The scaffold sequence may have structures such as a lower stem, bulge, upper stem, nexus, and / or hairpin. In some embodiments, the scaffold sequence within gRNA may consist of approximately 90 to 120 nucleic acids.

[0104] Zinc finger nuclease In some embodiments, nucleases are zinc finger nucleases (ZFNs). ZFNs typically contain a zinc finger DNA-binding domain and a nuclease domain. Generally, a ZFN contains two zinc finger arrays (ZFAs), each fused to a single subunit of a nonspecific endonuclease, such as a nuclease domain derived from the FokI enzyme, which becomes active by dimerization. Typically, a single ZFA consists of three or four zinc finger domains, each designed to recognize a specific nucleotide triplet (GGC, GAT, etc.). Thus, a ZFN composed of two "three-finger" ZFAs can recognize an 18-base pair target site (i.e., a recognition sequence). The 18-base pair recognition sequence is usually a unique sequence, even within large genomes such as the human and plant genomes. ZFNs induce colocalization and dimerization of two FokI nuclease monomers, thereby generating functional site-specific endonucleases that can target specific loci (e.g., genes, promoters, or enhancers).

[0105] Zinc finger nucleases useful in the methods disclosed herein include known ones and ZFNs engineered to have specificity to one or more target sites (e.g., nucleotide sequences of promoters or enhancers) as described herein. The zinc finger domain can be used to design polypeptides that specifically bind to selected polynucleotide recognition sequences within target sites in the host cell genome. ZFNs may include engineered DNA-binding zinc finger domains linked to a nonspecific endonuclease domain, such as a nuclease domain derived from a type II endonuclease like HO or FokI. In some examples, the zinc finger DNA-binding domain may be fused to a site-specific recombinase, transposase, or derivative thereof that retains DNA nicking and / or cleavage activity.

[0106] In some embodiments, additional functional moieties, non-limitingly containing transcriptional activator domains (such as VP16, VP48, VP64, and VP160) or transcriptional repressor domains (such as KRAB), may be fused to the zinc finger binding domain. In one embodiment, the zinc finger nuclease is engineered to contain a transcriptional activator domain selected from VP16, VP48, VP64, or VP160. In one embodiment, the zinc finger nuclease is engineered to contain 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-γ, and SET9. For example, engineered zinc finger transcription activators that interact with the promoter region of the γ-globulin gene have been shown to enhance fetal hemoglobin production in primary adult erythroblasts (Wilber et al., Blood, 115(15): 3033-3041). Other zinc finger transcription factors containing multiple finger moieties are also known in the art, including those disclosed by Beerli and Barbas (see Nature Technology, (2002) 20:135-141).

[0107] Each zinc finger domain recognizes three consecutive base pairs in target DNA. For example, three finger domains recognize a sequence of nine consecutive nucleotides, but because nuclease dimerization is required, two sets of zinc finger triplets are used to bind to an 18-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).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; U.S. Patent Application Publication No. 2003 / 0108880; also see U.S. Patents No. 6,140,466, No. 6,511,808, and No. 6,453,242. Useful zinc finger nucleases also include those listed in WO03 / 080809, WO05 / 014791, WO05 / 084190, WO08 / 021207, WO09 / 042186, WO09 / 054985, and WO10 / 065123.

[0108] In some embodiments, the ZFN comprises a fusion protein having a cleavage domain of an IIS-type restriction endonuclease fused to an engineered zinc finger-binding domain, wherein the binding domain further comprises one or more transcription activators. In some embodiments, the IIS-type restriction endonuclease is selected from HO endonuclease or FokI endonuclease. In some embodiments, the zinc finger-binding domain comprises three, four, five, or six 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., promoters or enhancers of SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A, and PAX6). In one embodiment, one or more transcription activators are selected from VP16, VP48, VP64, or VP160. Generally, the DNA-binding domain of a ZFN contains 3 to 6 distinct zinc finger repeats and can recognize 9 to 18 consecutive nucleotides. Each ZFN can be designed to target a specific target site within the host cell genome, such as a promoter sequence, enhancer sequence, or exon / intron within a gene.

[0109] TALEN In some embodiments of the methods and compositions described herein, the nuclease is a TALEN. TAL effectors (TALEs) are proteins secreted by bacteria of the genus Xanthomonas, and play a crucial role in inducing disease or defense mechanisms by binding to host DNA and activating effector-specific host genes. See, for example, 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. TALEN contains a TAL effector DNA-binding domain fused to a DNA-cleaving domain. DNA-binding domains interact with DNA in a sequence-specific manner via one or more tandem repeat domains. Repeat sequences typically contain 33–34 highly conserved amino acids, with the 12th and 13th amino acids differing. These two positions, called repeat variable duos (RVDs), are highly variable and show a strong correlation with individual nucleotide recognition (Boch et al., (2009) Science 326(5959):1509-12 and Moscow and Bogdanove, (2009) 326(5959):1501). This relationship between amino acid sequences and DNA recognition sequences has made it possible to artificially create specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs.

[0110] The TAL effector DNA-binding domain can be manipulated to bind to a target DNA sequence and can be fused to a nuclease domain, such as an IIS-type restriction endonuclease like FokI (see, e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160). In some embodiments, the nuclease domain may contain one or more mutations (e.g., FokI variants) that enhance cleavage specificity (see Doyon et al., (2011) Nature Methods, 8 (1): 74-9) and cleavage activity (see Guo et al., (2010) Journal of Molecular Biology, 400 (1): 96-107). Other useful endonucleases that can be used as nuclease domains include, but are not limited to, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. In some embodiments, a TALEN may include a TAL effector DNA-binding domain comprising multiple TAL effector repeat sequences that bind to a specific nucleotide sequence (i.e., a recognition sequence) in target DNA. While not to be construed as limiting, TALENs useful in the methods provided herein include those described in WO10 / 079430 and U.S. Patent Application Publication 2011 / 0145940.

[0111] In some embodiments, the TAL effector DNA-binding domain may include 10 or more DNA-binding repeats, and preferably 15 or more. In some embodiments, each DNA-binding repeat includes an RVD that determines the recognition of a base pair in the target DNA, where each DNA-binding repeat is responsible for the recognition of one base pair in the target DNA. In some embodiments, the RVD includes one or more of the following: 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 at the second position of the RVD), HG for recognizing T, H* for recognizing T (where * represents a gap at 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.

[0112] In some embodiments, TALENs are engineered so that the TAL effector contains one or more transcription activator domains (e.g., VP16, VP48, VP64, or VP160). For example, engineered TAL effectors having a transcription activator domain at the C-terminus have been shown to regulate the transcription of the 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 Perez-Pinera et al., who demonstrated the regulation of the IL1RN, KLK3, CEACAM5, and ERBB2 genes in human 293T cells using TALE-TFs (Nature Methods, (2013)). This also includes those disclosed in 10(3):239-242). In some embodiments, one or more transcription activator domains are located adjacent to the nuclear localization signal (NLS) present at the C-terminus of the TAL effector. In another embodiment, the TALE-TF can bind to a neighboring site upstream or downstream of the transcription start site (TSS) of a target gene. In one embodiment, the TAL effector comprises a transcription activator domain selected from VP16, VP48, VP64, or VP160. In another embodiment, the TAL effector comprises a transcription 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-γ, and SET9.

[0113] In some embodiments, TALEN comprises a TAL effector DNA-binding domain fused to a DNA cleavage domain, where the TAL effector comprises a transcription activator. In some embodiments, the DNA cleavage domain is an IIS-type restriction endonuclease selected from HO endonucleases or FokI endonucleases. In some embodiments, the TAL effector DNA-binding domain specifically binds to a recognition sequence corresponding to a promoter or enhancer region disclosed herein (e.g., promoters or enhancers of SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A, and PAX6).

[0114] H Expression vector for selected MHC alleles In some embodiments, the engineered mammalian dendritic cells described herein include one or more expression vectors for expressing exogenous MHC class II alleles. A wide variety of expression vectors may be used, including retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, plasmids, or transposons. Viral vectors that may be used include, for example, vectors based on HIV, SV40, EBV, HSV, or BPV. The expression vectors may be designed as replication-deficient viruses such that the viral vector lacks one or more functions essential for viral genome replication or synthesis and assembly of viral particles. Many replication-deficient viruses currently available can carry large therapeutic genes, effectively transduce various types of cells, and achieve long-term and stable expression of genes of interest.

[0115] Lentiviruses are a subset of retroviruses commonly used in research. Lentiviruses can transduce both dividing and non-dividing cells without eliciting a significant immune response. These viruses also stably integrate into the host genome, enabling long-term transgene expression. A common lentivirus is the human immunodeficiency virus (HIV), which uses envelope glycoproteins from other viruses to target a wide range of cell types.

[0116] One of the safety features of lentiviruses is that the components necessary to create infectious viral particles (virions) are usually separated across multiple plasmids. For example, an infectious viral particle may contain plasmids for the components of the viral capsid and envelope (usually called packaging plasmids and envelope plasmids) as well as plasmids that encode the viral genome (typically called transfer plasmids). Common lentiviral packaging plasmids and lentiviral envelope plasmids that may 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 may 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, pLionll, pSLIK-Hygro, and pInducer10-mir-RUP-PheS.

[0117] There are several approaches to constructing 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 can be purchased from commercial suppliers. Generally, lentiviral vector production involves multiple steps, including plasmid development and fabrication, cell proliferation, plasmid transfection, viral vector fabrication, purification, loading, and finishing. (See, for example, www.addgene.org / viral-vectors / lentivirus / ; www.thermofisher.com / us / en / home / clinical / cell-gene-therapy / gene-therapy / lv-production-workflow.html).

[0118] Lentiviral vectors may be designed to simultaneously express one or more genes of interest. Various molecular strategies are available, including the use of multiple promoters, splicing signals, gene fusion, cleavage factors, and multicistronic vectors. (See, for example, the review by Shaimardanova et al., “Production and application of multicistronic constructs for various human disease therapies.” Pharmaceutics 2019, 11, 580).

[0119] 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 microbubbles, magnetofection, and inorganic molecules.

[0120] I Composition In one aspect, the present disclosure provides a composition comprising engineered mammalian dendritic cells. As described herein, the engineered mammalian dendritic cells comprise one or more exogenous alleles for MHC class II genes.

[0121] In some aspects, the composition contains 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, and a small number of The composition contains 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 composition contains at least 1,000,000 cells. In some embodiments, the composition contains at least 20,000,000 cells.

[0122] In some embodiments, the composition contains up to 10,000 cells, up to 100,000 cells, up to 1,000,000 cells, up to 1,250,000 cells, up to 1,500,000 cells, up to 2,000,000 cells, up to 2,500,000 cells, up to 3,000,000 cells, up to 3,500,000 cells, up to 4,000,000 cells, up to 4,500,000 cells, and up to The composition contains up to 5,000,000 cells, up to 10,000,000 cells, up to 12,500,000 cells, up to 15,000,000 cells, up to 20,000,000 cells, up to 25,000,000 cells, up to 30,000,000 cells, up to 35,000,000 cells, up to 40,000,000 cells, up to 45,000,000 cells, or up to 50,000,000 cells. In some embodiments, the composition contains up to 20,000,000 cells. In some embodiments, the composition contains up to 40,000,000 cells.

[0123] In some embodiments, the composition contains approximately 1,000,000 to approximately 50,000,000 cells, approximately 5,000,000 to approximately 35,000,000 cells, approximately 10,000,000 to approximately 25,000,000 cells, approximately 15,000,000 to approximately 20,000,000 cells, or approximately 35,000,000 to approximately 40,000,000 cells. In some embodiments, the composition contains approximately 1,000,000 cells. In some embodiments, the composition contains approximately 20,000,000 cells. In some embodiments, the composition contains approximately 40,000,000 cells.

[0124] In another aspect, this disclosure provides pharmaceutical compositions. In some embodiments, a pharmaceutical composition comprises any of the compositions described herein and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition may comprise engineered mammalian dendritic cells or cell lines comprising at least one, two, three, four, five, or more exogenous alleles encoding at least one MHC class II gene. In some embodiments, the engineered mammalian dendritic cells or cell lines may also comprise at least one, two, three, four, five, or more exogenous alleles encoding at least one MHC class I gene. In some embodiments, the engineered mammalian dendritic cells or cell lines may further comprise one or more costimulatory molecules, heterologous antigens, and / or cytokines as described herein. At least one, two, three, four, five, or more recombinant polynucleotides may comprise, for example, heterologous sequences encoding costimulatory molecules, heterologous antigens, cytokines, or 2A splicing peptides. Therefore, recombinant polynucleotides encoding one or more MHC alleles, costimulatory molecules, heteroantigens, and / or cytokines may be separated by sequences encoding 2A splicing peptides (e.g., T2A, P2A, E2A). Typically, at least one, two, three, four, five, or more exogenous alleles are cloned into an expression vector (e.g., a replication-deficient lentiviral vector) for the synthesis of MHC alleles, costimulatory molecules, heteroantigens, and / or cytokines, and introduced into engineered mammalian dendritic cells or cell lines. Thus, engineered mammalian dendritic cells or cell lines provided in a pharmaceutical composition may have at least one, two, three, four, five, or more expression vectors, each expression vector containing at least one, two, three, four, five, or more exogenous alleles encoding MHC alleles, costimulatory molecules, heteroantigens, and / or cytokines.

[0125] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant, interferon α (e.g., IFN-α2a or IFN-α2b), and / or interferon λ family members (e.g., interferon λ1 (IFNλ1(IL-29)), interferon λ2 (IFNλ2(IL-28A)), interferon λ3 (IFNλ1(IL-28B)), interferon λ4 (IFNλ4)). In some embodiments, interferon α (e.g., IFN-α2a or IFN-α2b) is expressed in engineered mammalian dendritic cells as described herein by a vector containing a polynucleotide sequence of the IFNA2 gene. In some embodiments, interferon α is exogenously provided pegylated IFN-α2a. In some embodiments, the pharmaceutical composition further comprises one or more excipients. In some embodiments, the pharmaceutical composition further comprises the cryopreservation medium CryoStor CS10, CryoStor CS2, or CryoStor CS5. In certain embodiments, the pharmaceutical composition comprises cells cryopreserved in cryopreservation media CryoStor CS10, CryoStor CS2, or CryoStor CS5.

[0126] In some embodiments, the pharmaceutical composition is formulated in dosage forms containing a total number of engineered mammalian dendritic cells per dose for administration to a subject requiring it. In some embodiments, the pharmaceutical composition is formulated as a “ready-made” product for self-administration to a subject requiring it. In some embodiments, the pharmaceutical composition contains 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. The composition may contain 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 contains at least 1,000,000 cells. In some embodiments, the pharmaceutical composition contains at least 20,000,000 cells.

[0127] In some embodiments, the pharmaceutical composition contains up to 10,000 cells, up to 100,000 cells, up to 1,000,000 cells, up to 1,250,000 cells, up to 1,500,000 cells, up to 2,000,000 cells, up to 2,500,000 cells, up to 3,000,000 cells, up to 3,500,000 cells, up to 4,000,000 cells, up to 4,500,000 cells, The composition contains up to 5,000,000 cells, up to 10,000,000 cells, up to 12,500,000 cells, up to 15,000,000 cells, up to 20,000,000 cells, up to 25,000,000 cells, up to 30,000,000 cells, up to 35,000,000 cells, up to 40,000,000 cells, up to 45,000,000 cells, or up to 50,000,000 cells. In some embodiments, the pharmaceutical composition contains up to 20,000,000 cells. In some embodiments, the pharmaceutical composition contains up to 40,000,000 cells.

[0128] In some embodiments, the pharmaceutical composition contains approximately 1,000,000 to approximately 50,000,000 cells, approximately 5,000,000 to approximately 35,000,000 cells, approximately 10,000,000 to approximately 25,000,000 cells, approximately 15,000,000 to approximately 20,000,000 cells, or approximately 35,000,000 to approximately 40,000,000 cells. In some embodiments, the pharmaceutical composition contains approximately 1,000,000 cells. In some embodiments, the pharmaceutical composition contains approximately 20,000,000 cells. In some embodiments, the pharmaceutical composition contains approximately 40,000,000 cells.

[0129] In some embodiments, pharmaceutical compositions are formulated in the form of suspensions. Formulation of pharmaceutical compositions is generally known in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990)). Prevention of microbial contamination can be achieved by adding one or more of various antimicrobial and antifungal agents. In certain embodiments, the pharmaceutical composition is a liquid formulation containing cells resuspended in Ringer's lactate solution.

[0130] Suitable pharmaceutical forms for administration include sterile aqueous solutions or dispersions, and sterile powders for preparing sterile injection solutions or dispersions at the time of use. Typical carriers include solvents or dispersion media, such as aqueous buffer solutions (i.e., biocompatible buffers; non-limiting examples include Ringer's lactate solution and CryoStor cryopreservation media (e.g., CS2, CS5, and CS10 containing 2%, 5%, and 10% DMSO, respectively; available from BioLife Solutions (Bothell, WA))), ethanol, polyols, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils.

[0131] Sterilization can be carried out using techniques known in the art, including but not limited to the addition of antimicrobial or antifungal agents, such as parabens, chlorobutanol, sorbic acid, or thimerosal. Furthermore, isotonic agents such as sugars or sodium chloride may be incorporated into the composition.

[0132] The preparation of a sterile injection solution containing manipulated mammalian dendritic cells and / or other compositions according to the present disclosure can be carried out by mixing the required amount of the compound in a suitable solvent containing the various components listed above, as needed, and then sterilizing it. To obtain a sterile powder, the above sterile solution can be vacuum-dried or freeze-dried as needed.

[0133] In some embodiments, the engineered mammalian dendritic cells and / or other compositions provided herein are formulated for administration in unit dosage forms, e.g., intradermal injection, intralymphatic injection, oral, nasal, topical, or parental administration, for ease of administration and uniformity of dosage. As used herein, unit dosage form means a physically distinct unit suitable as a unit dose to a subject to be treated, e.g., a human or other mammal, each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect in conjunction with the necessary pharmaceutical carrier. In some examples, higher concentration dosage forms may be prepared, and then lower dilute unit dosage forms may be made from them. Thus, higher concentration dosage forms contain substantially larger amounts, e.g., at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, or more, of the engineered mammalian dendritic cells and / or other compositions.

[0134] In some embodiments, the engineered mammalian dendritic cells and / or other compositions provided herein are formulated for administration, for example, for one or more doses over a period of time. In some embodiments, the engineered mammalian dendritic cells and / or other compositions are formulated for weekly, bi-weekly, tri-weekly, quar-weekly, quin-weekly, or quin-weekly administration. In some embodiments, the engineered mammalian dendritic cells and / or other compositions are formulated for monthly, bi-monthly, tri-monthly, quar-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, quin-monthly, or quin-monthly administration.

[0135] The dosage is, for example, approximately 50,000 to approximately 50,000,000 (for example, approximately 50,000, approximately 60,000, approximately 70,000, approximately 80,000, approximately 90,000, approximately 100,000, approximately 110,000, approximately 120,000, approximately 130,000, approximately 140,000, approximately 150,000, approximately 160,000, approximately 170,000, approximately 180,000, approximately 190,000, approximately 200,000, approximately 250,000, approximately 300 ,000 pieces, about 350,000 pieces, about 400,000 pieces, about 450,000 pieces, about 500,000 pieces, about 550,000 pieces, about 600,000 pieces, about 650,000 pieces, about 700,000 pieces, about 750,000 pieces, about 800, 000 pieces, about 850,000 pieces, about 900,000 pieces, about 950,000 pieces, about 1,000,000 pieces, about 1,500,000 pieces, about 2,000,000 pieces, about 2,500,000 pieces, about 3,000,000 pieces, about 3,500 pieces ,000 pieces, about 4,000,000 pieces, about 4,500,000 pieces, about 5,000,000 pieces, about 5,500,000 pieces, about 6,000,000 pieces, about 6,500,000 pieces, about 7,000,000 pieces, about 7,500,000 pieces , about 8,000,000 pieces, about 8,500,000 pieces, about 9,000,000 pieces, about 9,500,000 pieces, about 10,000,000 pieces, about 11,000,000 pieces, about 12,000,000 pieces, about 13,000,000 pieces, The dose may contain approximately 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 manipulated mammalian dendritic cells. In some embodiments, the dose may contain approximately 1,000,000 manipulated mammalian dendritic cells. In some embodiments, the dose may contain approximately 5,000,000 manipulated mammalian dendritic cells. In some embodiments, the dose may contain approximately 10,000,000 manipulated mammalian dendritic cells.In some embodiments, the dose may contain approximately 20,000,000 manipulated mammalian dendritic cells.

[0136] The dosage is also, for example, at least about 5,000,000 to about 100,000,000 (for example, about 5,000,000, about 6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, about 10,000,000, about 15,000,000, about 20,000,000, about 25,000,000, about 30,000,000, about 35,000,000, about 40,000,000) The dose may contain manipulated mammalian dendritic cells in quantities of approximately 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. In some embodiments, the dose may contain at least approximately 1,000,000 manipulated mammalian dendritic cells. In some embodiments, the dose may contain at least approximately 5,000,000 manipulated mammalian dendritic cells. In some embodiments, the dose may contain at least about 10,000,000 engineered mammalian dendritic cells. In some embodiments, the dose may contain at least about 20,000,000 engineered mammalian dendritic cells.

[0137] The dosage can be alternatively, for example, at least about 100,000,000 to about 1,000,000,000 (e.g., about 100,000,000, about 150,000,000, about 200,000,000, about 250,000,000, about 300,000,000, about 350,000,000, about 400,000,000, about 450,000,000, about 500,000,000) It may also contain manipulated mammalian dendritic cells (approximately 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).

[0138] Methods for preparing such dosage forms are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, above). Typically, these dosage forms contain conventional pharmaceutical carriers or excipients and may additionally contain other pharmaceutical substances, carriers, adjuvants, diluents, tissue permeability enhancers, and solubilizers. Appropriate excipients can be tailored to individual dosage forms and routes of administration by methods well known in the art (see, for example, Remington's Pharmaceutical Sciences, above).

[0139] Suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, arginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and polyacrylic acid, such as Carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981. The dosage form may additionally contain lubricants such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers; suspending agents; preservatives such as methyl hydroxybenzoate, ethyl hydroxybenzoate, and propyl hydroxybenzoate (i.e., parabens); pH adjusters such as inorganic and organic acids and bases; sweeteners; and flavoring agents. The dosage form may also include biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0140] In some embodiments, the therapeutically effective dose may further include other components, such as anti-allergic agents, including antihistamines, steroids, bronchodilators, leukotriene stabilizers, and mast cell stabilizers. Such anti-allergic agents are well known in the art.

[0141] J Methods for treating cancer Methods for immunotherapy using hemi-allogeneic dendritic cells This disclosure provides a method for immunotherapy using hemoallogeneic dendritic cells in a subject. In some embodiments, the method includes administering a therapeutically effective amount of a pharmaceutical composition of the Disclosure described herein (e.g., a pharmaceutical composition comprising manipulated mammalian dendritic cells of the Disclosure) to a subject.

[0142] In some embodiments, the method includes, prior to the administration step, (i) obtaining an MHC class II allele profile by genotyping several MHC class II genes in a biological sample derived from a subject, and (ii) selecting engineered mammalian dendritic cells for administration to the subject, wherein the engineered mammalian dendritic cells contain one or more mismatches with the subject's MHC class II allele profile. In some embodiments, the subject is human. In some embodiments, the method for immunotherapy using hemi-allogeneic dendritic cells is for the treatment of human cancer. In some examples, the engineered human dendritic cells or a group of engineered human dendritic cells for cancer treatment contain a tumor-specific antigen or a fragment thereof.

[0143] Methods for immunotherapy using autologous dendritic cells This disclosure also provides a method for immunotherapy using autologous dendritic cells in a subject. As described herein, the method includes the step of administering a therapeutically effective amount of a pharmaceutical composition of the Disclosure (e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the Disclosure) to a subject, wherein the engineered mammalian dendritic cells are derived from the primary cells of the subject. In some embodiments, primary cells are primary immune cells, which include, but are not limited to, dendritic cells, monocytes, macrophages, B cells, T cells, nature killer (NK) cells, and neutrophils. A specific example of primary immune cells is dendritic cells.

[0144] In some embodiments, a method for immunotherapy using autologous dendritic cells further comprises, prior to the administration step, (i) obtaining primary immune cells or a plurality of primary immune cells from a subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cells in order to determine the endogenous MHC class II allele profile; and (iii) manipulating the primary immune cells into engineered mammalian dendritic cells by (a) introducing one or more exogenous MHC class II alleles containing at least one mismatch with the endogenous MHC class II allele profile of the subject into the primary immune cells; and (b) introducing antigens or fragments thereof of cancer or pathogens affecting the subject into the primary immune cells. In some embodiments, the manipulating into mammalian dendritic cells further comprises (c) incubating the primary immune cells with lipopolysaccharide (LPS), interferon-γ (IFN-γ), or a combination of LPS and IFN-γ.

[0145] Treg inhibitors In some embodiments, this disclosure provides methods for enhancing or inducing an anti-cancer response in a subject by using a regulatory T cell inhibitor (Treg inhibitor or Treg activator) that inhibits or reduces the activity or function of Tregs in order to enhance the efficacy of the cancer vaccines described herein. The method comprises the steps of administering an effective amount of a Treg activator to a subject, wherein the Treg activator reduces the activity or function of Tregs, and administering an effective amount of a pharmaceutical composition comprising engineered mammalian dendritic cells disclosed herein. In some examples, the Treg activator and the pharmaceutical composition are administered to the subject simultaneously. In other examples, the Treg activator is administered to the subject before the pharmaceutical composition is administered. In yet another example, the Treg activator is administered to the subject after the pharmaceutical composition is administered. In certain embodiments, the Treg activator is administered to the subject one hour, several hours, one day, several days, one week, several weeks, one month, or several months after the administration of the pharmaceutical composition.

[0146] Regulatory T cell inhibitors (Treg inhibitors or Treg activators) disclosed herein may be any Treg target substances, including but not limited to any compounds, small molecules, toxins, polynucleotides (such as aptamers, RNAi, siRNA, or antisense oligonucleotides), polypeptides, proteins (such as antibodies), fusion proteins, drug conjugates, and chemotherapeutic agents, that (i) inhibit or reduce the function of Treg cells and / or (ii) deplete or reduce the regulatory T cell population.

[0147] In some embodiments, the Treg activator is selected from the group consisting of antibodies, small molecules, antibody-drug conjugates, immunotoxins, peptide-drug conjugates, peptides, small interfering RNAs, siRNA conjugates, chemotherapeutic agents, and any derivatives, fragments, or fusions thereof. Examples of Treg-activating substances include, but are not limited to, 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, and 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 entirety of which is incorporated herein by reference.

[0148] In certain embodiments, the Treg activator may include an antibody or fragment thereof that specifically binds to regulatory T cell surface proteins. The antibody contained in the Treg activator may target surface proteins of Treg cells, such as CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), cytotoxic T lymphocyte-associated antigen 4 (CTLA4), CCR4, CCR8, FOXP3, LAG3, CD103, NRP-1, glucocorticoid-inducible TNF receptor (GITR), galectin-1, TNFR2, or TGF-βR1. In some embodiments, the Treg activator may 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 the CD25 or interleukin-2 receptor α subunit (IL-2Rα) and is overexpressed by activated T cells. Zenapax is an immunosuppressive humanized IgG1 monoclonal antibody that binds to the human high-affinity IL-2 receptor CD25 subunit expressed on the surface of activated lymphocytes. MDX-010 is a monoclonal antibody that targets CTLA4.

[0149] In certain embodiments, the Treg activator may include an antibody-drug conjugate. As disclosed herein, the antibody or a fragment thereof may further include a radionuclide or toxic portion so that the antibody can kill regulatory T cells. In some embodiments, radionuclides suitable for use in this disclosure may include those having radioactive properties suitable for in situ removal of targeted Tregs without excessively exposing surrounding cells and tissues to damaging levels of irradiation. Ideal radionuclides for use in such therapeutic compositions are relatively short-lived alpha, gamma, or beta emitters that emit enough gamma rays to cause localized destruction. Non-limiting examples of radionuclides include lutetium-177, iodine-131, iodine-125, and phosphorus-32 (γ-emitting); actinium-225, astatine-211, and bismuth-212 and bismuth-213 (α-emitting); iodine-123, copper-64, iridium-192, osmium-194, rhodium-105, rhodium-186, samarium-153, and yttrium-88, yttrium-90, and yttrium-91.

[0150] In certain embodiments, the Treg activator may include a fusion protein. In some embodiments, the fusion protein may include a targeting moiety and a toxic moiety. The targeting moiety may include a ligand or a portion thereof for a surface protein of regulatory T cells. The ligand may be, for example, IL2, T cell receptor (TCR), MHCII, CD80, CD86, TARC, CCL17, CKLF1, CCL1, TCA-3, eotaxin, TER-1, E-cadherin, VEGF, semaphorin 3a, CD134, CD31, CD62, CD38L, or glucocorticoid-inducible TNF receptor ligand (GITRL). The toxic moiety may be, for example, lectin, lysine, abrin, biscumin, modesin, diphtheria toxin, cholera toxin, geronin, Pseudomonas exotoxin, Shigella toxin, botulinum toxin, tetanus toxin, calichemycin, or pokeweed antiviral protein.

[0151] In certain embodiments, Treg activators may include immunotoxins. Immunotoxins can be immune conjugates that induce target cell death by combining antibodies with target-specific high-affinity binding activity with other molecules such as radioisotopes, chemicals, siRNAs, and cytotoxic proteins. In some embodiments, immunotoxins include denileukin difutitox (Ontak), tagraxofusp (Elzonris), moxetumomab pasdotox (Lumoxiti), or any derivatives or combinations thereof.

[0152] In certain embodiments, the Treg activator may include a peptide-drug conjugate (PDC). The PDC contains a peptide linked to a payload. The peptide in the PDC may be specific to the target cell and may induce receptor-mediated endocytosis of the conjugate. The payload may be a highly toxic drug such as maytansine, a camptothecin derivative, auristatin, or doxorubicin.

[0153] In certain embodiments, Treg activators may include small interfering RNA (siRNA). siRNA is a 21-23 nucleotide long double-stranded RNA (dsRNA) molecule that specifically induces RNA interference (RNAi), a post-transcriptional method for silencing gene expression. In certain embodiments, siRNA forms complexes with biomolecules such as lipophilic molecules, antibodies, aptamers, ligands, peptides, or polymers. Aptamers are single-stranded oligonucleotides that recognize targets through unique three-dimensional complementarity. In some embodiments, Treg activators may include siRNA conjugates such as CTLA4apt-STAT3 siRNA, NPsiCTLA-4, or hybrid SNPs. CTLA4apt-STAT3 siRNA is an siRNA conjugate in which a CTLA4-binding RNA aptamer is linked to mouse STAT3 siRNA. siCTLA-4 This is a nanostructured material-siRNA conjugate in which an siRNA targeting CTLA-4 mRNA is surrounded by nanoparticles composed of PEG5k-PLA11k and BHEM-Chol. The hybrid SNP is a spherical nucleotide nanoparticle (SNP) in which a CTLA-4-siRNA aptamer (cSNP) and a PD-1 siRNA (pSNP) are packed inside a nanoparticle containing a core amphiphilic polymer PLGA-SS-PEG and a cationic lipid DOTAP.

[0154] According to the methods of this disclosure, Treg agonists modulate regulatory T cells by reducing the activity or function of Tregs after administration to a target; or, Treg agonists bound to toxic moieties can kill or eliminate regulatory T cells. Administration of Treg agonists or derivatives can interfere with the action of their targets (e.g., Treg cell surface markers). Thus, Treg agonists can reduce the suppression of immune system activation and reduce the prevention of autoreactivity. Such reductions can be measured by techniques established in the art. See, for example, 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 assay methods used for detecting T cell responses include delayed-type hypersensitivity responses, in vitro T cell proliferation responses (e.g., measured using radioactive nucleotide uptake), library screening, expression arrays, T cell cytokine responses (e.g., measured by ELISA or other relevant immunoassays or RT-PCR of specific cytokine mRNAs), as well as any other assay methods established in the art for measuring B cell and / or T cell immune responses in a subject. Methods for detecting immune responses may include, but are not limited to, antibody detection assays, e.g., EIA (enzyme-linked immunoassay), ELISA (enzyme-linked immunosorbent assay), agglutination reactions, precipitation / coagulation reactions, immunoblotting (Western blot, dot / slot blot), radioimmunoassays, immunodiffusion assays (RIA), histochemical assays, immunofluorescence assays (FACS), chemiluminescence assays, library screening, expression arrays, etc.

[0155] Concomitant use with other active substances or therapies In some embodiments, the method further includes the step of intravenously administering cyclophosphamide to the subject one or more times at least one day, at least two days, at least three days, at least four days, at least five days, or earlier than the time of administration of the pharmaceutical composition described herein. In some embodiments, the cyclophosphamide is administered at least about two to three days before the time of administration of the pharmaceutical composition described herein. In some embodiments, about 100 mg / m² 2 , about 150mg / m 2 , about 200mg / m 2 , about 250mg / m 2 , about 300mg / m 2 , or approximately 450 mg / m² 2 Low doses of cyclophosphamide, such as those mentioned above, are administered to the patients.

[0156] In some embodiments, the method further includes the step of intradermally administering interferon-α-2b (IFN-α2b), IFN-α2a, or pegylated IFN-α2a to a subject one or more times at the inoculation site of the pharmaceutical composition described herein. In some embodiments, the method further includes the step of intradermally administering IFN-α2b, IFN-α2a, or pegylated IFN-α2a to a subject one or more times within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours after administering the pharmaceutical composition described herein to the subject. In some embodiments, the method further includes the step of intradermally administering IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject one or more times about 1 to 4 hours, about 2 to 6 hours, about 8 to 12 hours, about 10 to 24 hours, about 20 to 48 hours, or about 60 to 72 hours after administering the pharmaceutical composition described herein to the subject. In some embodiments, the method further includes the step of intradermally administering IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject one or more times within 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 45 hours, 50 hours, 60 hours, 72 hours, or 84 hours after administering the pharmaceutical composition to the subject. In some embodiments, the method further includes the step of intradermally administering IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject one or more times within 1, 2, 3, 4, 5, or 6 days after administering the pharmaceutical composition to the subject. In some embodiments, the method further includes the step of intradermally administering IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject one or more times within about 1 to 6 days, about 2 to 3 days, or about 3 to 5 days after administering the pharmaceutical composition to the subject. In some embodiments, the method further includes the step of intradermally administering the first dose of IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject 1 to 4 hours after administering the pharmaceutical composition to the subject, and the second dose of IFN-α2b, IFN-α2a, or pegylated IFN-α2a to the subject within 1 to 3 days.In some embodiments, IFN-α2b is administered in low doses of approximately 1–20,000 IU, approximately 100–15,000 IU, approximately 5,000–12,000 IU, or approximately 9,000–11,000 IU. In some embodiments, IFN-α2b is administered in a dose of approximately 10,000 IU. In some embodiments, IFN-α2a or pegylated IFN-α2a is administered in low doses of approximately 0.01–0.1 micrograms (mcg), approximately 0.05–0.15 mcg, approximately 0.06–0.12 mcg, or approximately 0.09–0.11 mcg. In some embodiments, IFN-α2b is administered in a dose of approximately 0.1 mcg.

[0157] In some embodiments, the method further includes a step of administering one or more additional treatments. Examples of suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiotherapy, hormone therapy, differentiation inducers, and small molecule drugs. Those skilled in the art will be able to easily select an appropriate additional treatment.

[0158] The chemotherapeutic agents that may be used in this disclosure include alkylating agents (e.g., nitrogen mustards (e.g., mechloretamine, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozosin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), triazines (e.g., dacarbazine (DTIC), temozolomide), ethyleneimines (e.g., thiotepa, altoretamine (hexamethylmelamine)), platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, etc.) These include, but are not limited to, trexate (pemetrexed), anthracycline antitumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline antitumor antibiotics (e.g., actinomycin-D, bleomycin, mitomycin-C, mitoxantrone), mitotic inhibitors (e.g., taxanes (e.g., paclitaxel, docetaxel), epothirone (e.g., ixabepirone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine), estramustine, corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), L-asparaginase, bortezomib, and topoisomerase inhibitors. Combinations of chemotherapeutic agents may also be used.

[0159] Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerase, an enzyme that promotes changes in DNA structure by catalyzing the cleavage and rejoining of phosphodiester bonds in the DNA backbone. 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 increase in the number of single-strand and double-strand breaks, thereby reducing genomic stability. This reduction in genomic stability leads to apoptosis and cell death.

[0160] Topoisomerases are often classified into type I and type II topoisomerases. Type I topoisomerases are essential for easing DNA supercoil formation during DNA replication and transcription. Type I topoisomerases cause breaks in the single strands of DNA and rejoin these breaks to reconstruct a complete double-stranded DNA molecule. Examples of type I topoisomerase inhibitors include irinotecan, topotecan, camptothecin, and lamellarin D, all of which target type IB topoisomerases.

[0161] Type II topoisomerase inhibitors are broadly classified into topoisomerase toxins and topoisomerase inhibitors. Topoisomerase toxins target the topoisomerase-DNA complex, while topoisomerase inhibitors disrupt the metabolic turnover of the enzyme catalyst. Examples of type II topoisomerase inhibitors include amsacrin, etoposide, phosphate etoposide, teniposide, doxorubicin, and fluoroquinolones.

[0162] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some examples, 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, amsacrin, ellipticin, aurintricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, lamellarin D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), and combinations thereof. EGCG is an example of a plant-derived natural phenol that acts as a suitable topoisomerase inhibitor. In some examples, the topoisomerase inhibitor is doxorubicin.

[0163] Immunotherapy refers to any treatment that uses the target immune system to fight a disease (e.g., cancer). Methods of immunotherapy may aim to either enhance or suppress immune function. In cancer therapy, methods of immunotherapy typically aim to enhance or activate immune function. In some examples, immunotherapeutic agents include monoclonal antibodies that target specific types or parts of cancer cells. In some cases, antibodies are conjugated to parts such as drug molecules or radioactive materials. Antibodies may, in non-limiting examples, be derived from mice, chimeric, or humanized. Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, retifanlimab, rituximab, tocitumomab, and trastuzumab.

[0164] Immunotherapy agents can also include immune checkpoint inhibitors, which modulate the immune system's ability to distinguish between normal and "heterogeneous" cells. Programmed cell death protein 1 (PD-1) and protein death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors because disrupting the interaction between PD-1 and PD-L1 enhances the activity of immune cells against heterogeneous cells such as cancer cells. Examples of PD-1 inhibitors include pembrolizumab, retifanlimab, and nivolumab. An example of a PD-L1 inhibitor is atezolizumab.

[0165] Another immune checkpoint target for cancer treatment is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), a receptor that downregulates the immune cell response. Therefore, drugs that inhibit CTLA-4 can enhance immune function. One example of such a drug is ipilimumab, a monoclonal antibody that binds to and inhibits CTLA-4.

[0166] The term "radiotherapy" refers to the delivery of high-energy radiation to a target for the treatment of a disease (e.g., cancer). Radiation therapy can include the delivery of X-rays, gamma rays, and / or charged particles. Radiation therapy can be performed 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).

[0167] The term "hormone therapy drugs" can refer to hormone synthesis inhibitors, hormone receptor antagonists, or hormone replacement drugs. Hormone synthesis inhibitors 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 antiandrogens. Hormone replacement drugs include, but are not limited to, progestogens, androgens, estrogens, and somatostatin analogs. Aromatase inhibitors are used, for example, to treat breast cancer. Non-exclusive examples include letrozole, anastrozole, and aminoglutethimide. GnRH analogs may be used, for example, to induce chemical castration. Selective estrogen receptor antagonists commonly used for the treatment of breast cancer include tamoxifen, raloxifene, toremifene, and fulvestrant. Antiandrogen drugs, which bind to and inhibit androgen receptors, are commonly used to inhibit the effects of testosterone on the growth and survival of prostate cancer. Non-exclusive examples include flutamide, apalutamide, and bicalutamide.

[0168] The term "differentiation inducer" refers to any substance that promotes cell differentiation, which in cancerous contexts can lead to malignant cells becoming less like stem cells. A non-exclusive example of an anti-cancer differentiation inducer is retinoic acid.

[0169] Generally, small molecule drugs are pharmacological substances with a small molecular weight (i.e., less than approximately 900 daltons). Non-exclusive examples of small molecule drugs used to treat cancer include bortezomib (a proteasome inhibitor), imatinib (a tyrosine kinase inhibitor), celicyclib (a cyclin-dependent kinase inhibitor), and epacadostat (an indoleamine 2,3-dioxygenase (IDO1) inhibitor).

[0170] Administration In some embodiments, the method includes the step of intradermally administering an effective amount of the pharmaceutical composition to the upper back or thigh of the subject. These areas are selected because, without regard to any particular theory, they are less sensitive and therefore more tolerable to the patient due to the fewer nerves in the skin. Furthermore, nearby aspiration lymph nodes may carry antigens originating from breast tumors located in the upper and lower torso, which are common sites of breast cancer metastasis. The method may further include the step of administering the pharmaceutical composition to the subject at intervals of weekly, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. In some embodiments, the method includes the step of administering the pharmaceutical composition to the subject at intervals of monthly, every two months, every three months, every four months, every five months, every six months, every twelve months, every eighteen months, or every twenty-four months. In some embodiments, the method includes the step of administering the pharmaceutical composition to the subject for a period of at least six weeks, twelve weeks, twenty-four weeks, thirty-six weeks, forty-eight weeks, fifteen weeks, or longer. In some embodiments, the method includes administering a pharmaceutical composition to a subject for a period of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, or longer. In some embodiments, the method includes administering a pharmaceutical composition to a subject for a period of 6 weeks or less, 12 weeks or less, 24 weeks or less, 36 weeks or less, 48 ​​weeks or less, or 52 weeks or less. In some embodiments, the method includes administering a pharmaceutical composition to a subject for a period of 1 month or less, 2 months or less, 3 months or less, 4 months or less, 5 months or less, 6 months or less, 8 months or less, 10 months or less, or 12 months or less.

[0171] In some embodiments, the method includes administering an effective amount of a pharmaceutical composition to a target by oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intrafocal, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration. In some embodiments, the administration of an effective amount of a pharmaceutical composition is carried out by parenteral administration (e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial) or transmucosal administration (e.g., oral cavity, sublingual, palate, gingiva, nose, vagina, rectum, or percutaneous). In some embodiments, the method includes the use of liposomal formulations, intravenous injection, or transdermal patches.

[0172] Therapeutic agents such as manipulated mammalian dendritic cells, compositions, and pharmaceutical compositions disclosed herein can be administered using routes, dosages, and protocols readily known to those skilled in the art. Administration may be once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week. Therapeutic agents may be administered once, twice, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen times a week, or more times. In some cases, the engineered mammalian dendritic cells, compositions, and / or pharmaceutical compositions of the Disclosure may be administered as a single dose, concurrently (e.g., as separate doses or via different routes but in close proximity in time), or separately (e.g., as separate doses, including the same or different routes, but spaced about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer). If multiple doses are planned on the same day, or if a single dose includes one or more components (e.g., engineered mammalian dendritic cells and IFNα are administered separately), the doses may be, for example, once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, or more times per day.

[0173] In some cases, therapeutic administration can be given approximately once a week, once every two weeks, once every three weeks, or once a month. In other cases, therapeutic administration can be given approximately once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty times, twenty-one times, twenty-two times, twenty-two times, twenty-three times, twenty-four times, twenty-five times, twenty-six times, twenty-seven times, twenty-eight times, twenty-nine times, thirty times, or more times per month. Treatment may last for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer; approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer; or longer. At any time during treatment, the treatment plan may be adjusted as needed. For example, in response to the manipulated mammalian dendritic cells, composition, or pharmaceutical composition of this disclosure, different vaccines may be selected, one or more additional therapeutic substances or drugs may be selected, or any aspect of the treatment plan may be discontinued. It is believed that such decisions can be easily made by those skilled in the art, for example, by referring to the results of a comparison of allele profiles, changes in the activity and / or number of immune cells, and / or changes in the presence or levels of one or more biomarkers.

[0174] The engineered mammalian dendritic cells, compositions, and pharmaceutical compositions disclosed herein may be administered by any suitable route, including those described herein. In some embodiments, administration is performed by intradermal or intralymphatic injection. In some embodiments, a whole-cell cancer vaccine (e.g., including the engineered mammalian dendritic cells disclosed herein) is administered separately from interferon-alpha (IFNa). In some examples, IFNa is administered by topical injection. IFNa may be administered before and / or after the administration of the vaccine. The timing of individual injections may be any suitable interval, including those described herein.

[0175] Those skilled in the art will be able to easily determine the appropriate number of manipulated mammalian dendritic cells to be included in each dose. Doses can range, for example, from approximately 50,000 to approximately 50,000,000 (e.g., approximately 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, 30 0,000 pieces, about 350,000 pieces, about 400,000 pieces, about 450,000 pieces, about 500,000 pieces, about 550,000 pieces, about 600,000 pieces, about 650,000 pieces, about 700,000 pieces, about 750,000 pieces, about 800 ,000 pieces, about 850,000 pieces, about 900,000 pieces, about 950,000 pieces, about 1,000,000 pieces, about 1,500,000 pieces, about 2,000,000 pieces, about 2,500,000 pieces, about 3,000,000 pieces, about 3,50 0,000 pieces, about 4,000,000 pieces, about 4,500,000 pieces, about 5,000,000 pieces, about 5,500,000 pieces, about 6,000,000 pieces, about 6,500,000 pieces, about 7,000,000 pieces, about 7,500,000 pieces 8,000,000 pieces, 8,500,000 pieces, 9,000,000 pieces, 9,500,000 pieces, 10,000,000 pieces, 11,000,000 pieces, 12,000,000 pieces, 13,000,000 pieces The dose may contain approximately 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 manipulated mammalian dendritic cells. In some embodiments, the dose may contain approximately 1,000,000 manipulated mammalian dendritic cells. In some embodiments, the dose may contain approximately 5,000,000 manipulated mammalian dendritic cells.In some embodiments, the dose may contain approximately 10,000,000 engineered mammalian dendritic cells. In some embodiments, the dose may contain approximately 20,000,000 engineered mammalian dendritic cells.

[0176] The dosage is also, for example, at least about 5,000,000 to about 100,000,000 (for example, about 5,000,000, about 6,000,000, about 7,000,000, about 8,000,000, about 9,000,000, about 10,000,000, about 15,000,000, about 20,000,000, about 25,000,000, about 30,000,000, about 35,000,000, about 40,000,000) The cells may also contain manipulated mammalian dendritic cells in quantities of approximately 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.

[0177] The dosage can be alternatively, for example, at least about 100,000,000 units to about 1,000,000,000 units (e.g., about 100,000,000 units, about 150,000,000 units, about 200,000,000 units, about 250,000,000 units, about 300,000,000 units, about 350,000,000 units, about 400,000,000 units, about 450,000,000 units, about 500,000,000 units). It may also contain manipulated mammalian dendritic cells in quantities of approximately 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.

[0178] In some embodiments, manipulated mammalian dendritic cells are irradiated. The irradiation dose is, for example, about 2 to about 2,000 Gy (e.g., about 2 Gy, about 3 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 7 Gy, about 8 Gy, about 9 Gy, about 10 Gy, about 11 Gy, about 12 Gy, about 13 Gy, about 14 Gy, about 15 Gy, about 16 Gy, about 17 Gy, about 18 Gy, about 19 Gy, about 20 Gy). y, approximately 30 Gy, approximately 40 Gy, approximately 50 Gy, approximately 60 Gy, approximately 70 Gy, approximately 80 Gy, approximately 90 Gy, approximately 100 Gy, approximately 110 Gy, approximately 120 Gy, approximately 130 Gy, approximately 140 Gy, approximately 150 Gy, approximately 160 Gy, approximately 170 Gy, approximately 180 Gy, approximately 190 Gy, approximately 200 Gy, approximately 210 Gy, approximately 2 20 Gy, approximately 230 Gy, approximately 240 Gy, approximately 250 Gy, approximately 260 Gy, approximately 270 Gy, approximately 280 Gy, approximately 290 Gy, approximately 300 Gy, approximately 350 Gy, approximately 400 Gy, approximately 450 Gy, approximately 500 Gy, approximately 550 Gy, approximately 600 Gy, approximately 650 Gy, approximately 700 Gy, approximately 750 Gy, approximately 800 Gy The doses may be approximately 850 Gy, 900 Gy, 950 Gy, 1,000 Gy, 1,100 Gy, 1,200 Gy, 1,300 Gy, 1,400 Gy, 1,500 Gy, 1,600 Gy, 1,700 Gy, 1,800 Gy, 1,900 Gy, or 2,000 Gy. In certain embodiments, the manipulated mammalian dendritic cells are irradiated with a dose of approximately 100 Gy.

[0179] Vaccine effectiveness In some embodiments, the method for treating cancer of the present disclosure further includes the step of selecting a whole-cell cancer vaccine for a subject according to the method of the present disclosure as 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 progressing between stages. In some embodiments, the subject has precancerous lesions. In some embodiments, the subject does not have cancer.

[0180] In some embodiments, treating a subject includes inhibiting the growth of cancer cells, inhibiting the proliferation of cancer cells, inhibiting the migration of cancer cells, inhibiting the invasion of cancer cells, improving or resolving cancer symptoms, reducing the size (e.g., volume) of cancerous tumors, reducing the number of cancerous tumors, reducing the number of cancer cells, inducing necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death of cancer cells, or enhancing the therapeutic effect of a composition or pharmaceutical composition. In some embodiments, treating a subject extends survival. In some examples, overall survival is extended. In other examples, disease-free survival is extended. In some examples, progression-free survival is extended. In certain embodiments, treating a subject results in a reduction in tumor volume and / or an extension of survival.

[0181] In certain embodiments, treating the target enhances the therapeutic effects of anticancer therapies such as chemotherapeutic agents, immunotherapeutic agents, radiotherapy, hormone therapy, differentiation-inducing agents, and / or small molecule drugs.

[0182] In some embodiments, treating a subject reduces the presence or level of one or more heterogeneous antigens measured or detected in a sample obtained from the subject. In some embodiments, treating a subject increases the presence or level of one or more biomarkers measured or detected in a sample obtained from the subject. In certain embodiments, treating a subject does not change the presence or level of one or more biomarkers.

[0183] In some embodiments, treating a subject increases the activity and / or number of one or more types of immune cells. In some examples, the increase occurs in one cell type. In other examples, the increase occurs in multiple cell types. In some embodiments, the cells whose activity level and / or number increases are selected from the group consisting of peripheral blood mononuclear cells (PBMCs), lymphocytes (e.g., T lymphocytes, B lymphocytes, NK cells), monocytes, dendritic cells, macrophages, myeloid-derived suppressor cells (MDSCs), and combinations thereof. In certain embodiments, the activity level and / or number of immune cells are measured using the methods of this disclosure described herein.

[0184] In some embodiments, an increase in the activity and / or number of immune cells indicates that the subject needs to be given one or more additional doses of the pharmaceutical composition (e.g., comprising the manipulated mammalian dendritic cells of the present disclosure). In some examples, another vaccine is administered. Those skilled in the art will recognize that in some examples, an increase in the activity and / or number of immune cells occurs after the vaccine has been administered one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more times.

[0185] In some embodiments, samples are obtained from subjects. In other embodiments, samples are obtained from different subjects or populations of subjects. Samples can be used to select appropriate cancer vaccines of the Disclosure, to monitor responses to vaccine therapy, and / or to predict how subjects will respond to vaccine therapy. Samples obtained from different subjects and / or populations of subjects can be used, for example, to establish reference ranges to facilitate comparisons, which are part of the methods of the Disclosure. Samples can be obtained at any point in time, including before and / or after administration of the manipulated mammalian dendritic cells, pharmaceutical compositions, and / or other compositions of the Disclosure. In some embodiments, samples include whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal mucosa cells, tumor tissue, urine, fluids obtained from pleural fluid, hair, skin, or combinations thereof. In general, samples can include any biological fluid. For MHC typing, any type of cell, tissue, or biological fluid is suitable, as long as it contains a sufficient amount of DNA or RNA to enable typing. In some examples, samples include circulating tumor cells (CTCs). The sample may also consist of a combination of normal and cancer cells. In certain embodiments, the sample includes circulating tumor cells (CTCs). The sample can be obtained, for example, by biopsy, surgical excision, and / or fine-needle aspiration (FNA). The sample can be used to determine, measure, or detect MHC alleles, immune cell activity and / or numbers, and / or biomarkers, as described herein.

[0186] In some embodiments, the results of MHC typing (e.g., alleles present in the allele profile, results of allele profile comparison), measurements of immune cell activity and / or number, and / or measurements of the presence or level of biomarkers are recorded on tangible media. For example, the results of assays (e.g., alleles present in the allele profile, results of allele profile comparison, immune cell activity levels and / or number, presence or level (e.g., expression) of one or more biomarkers, and / or prognosis or diagnosis (e.g., whether cancer is present, whether the subject will respond to a vaccine, or whether the subject is responding to a vaccine) can be recorded on paper or electronic media (e.g., audiotape, computer disk, CD, flash drive, etc.).

[0187] In other embodiments, the method further includes a step of providing the patient (i.e., subject) with the results of an assay, prognosis, and / or diagnosis, as well as the results of treatment.

[0188] K Kit In another aspect, the Disclosure provides kits for treating subjects affected by cancer. In some embodiments, the kits comprise the engineered mammalian dendritic cell lines, compositions, and / or pharmaceutical compositions described herein. The kits are useful for treating any cancer, and some non-limiting examples of cancer 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, kidney cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancers described herein, including combinations thereof.

[0189] Materials and reagents for carrying out the various methods of this disclosure may be provided in kits to facilitate the execution of the methods. As used herein, the term “kit” includes combinations of articles that facilitate a process, assay, analysis, or operation. Specifically, the kits of this disclosure are useful in a wide range of applications, including, for example, diagnostic, prognostic, and therapeutic applications.

[0190] The kit may include chemical reagents and other components. Furthermore, the kit of this disclosure may, without limitation, include instructions for 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 the engineered mammalian dendritic cells or other compositions of this disclosure, apparatus and reagents for measuring the levels of biomarkers 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, samples suitable for use for standardization, samples suitable for use for normalization, and / or control samples. The kit of this disclosure may also be placed in a box with a lid, for example, for convenient storage and safe transport. For example, the kit may be stored and transported at room temperature on wet ice or with a coolant, or frozen in the gas phase of liquid nitrogen or in dry ice.

[0191] In some embodiments, the kit also includes negative and positive control samples for detecting MHC alleles, immune cell activity and / or number, and / or the presence or level of biomarkers. In some embodiments, the negative control sample is non-cancerous cells, tissue, or biological fluid obtained from a subject who is scheduled to receive or has already received treatment. In other embodiments, the negative control sample is obtained from an individual or group of individuals who do not have cancer. In other embodiments, the positive control sample is obtained from a subject or group of individuals who have cancer. In some embodiments, the kit includes a sample for preparing titration curves of one or more biomarkers in the sample, which helps to assess quantified levels of activity and / or number of one or more immune cells and / or biomarkers in the biological sample. [Examples]

[0192] IV. Example The present disclosure will be described in more detail with specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure. Those skilled in the art will readily recognize a variety of non-material parameters that can be changed or modified to produce essentially the same results.

[0193] Example 1: Mouse strain and dendritic cells This example demonstrates a method for generating syngeneic or semi-allogeneic bone marrow dendritic cells (BMDCs) from three different mouse strains.

[0194] Each strain of experimental mouse is homozygous and possesses a unique MHC haplotype. In this study, the following three mouse strains were used to generate bone marrow dendritic cells (BMDCs): C57BL / 6J(WT); B6.C-H2-K bm1 / ByJ(bm1); and B6(C)-H2-Ab1 bm12 / KhEgJ(bm12) (Figures 1A-1C). B6.C-H2-K bm1 / ByJ(bm1) mouse strain and B6(C)-H2-Ab1bm12 Both / KhEgJ(bm12) mouse strains originally originated from the C57BL / 6J(WT) mouse strain. B6.C-H2-K bm1 The / ByJ(bm1) mouse strain is MHC class I H2-K b It has a point mutation in the allele. B6(C)-H2-Ab1 bm12 The / KhEgJ(bm12) mouse strain is MHC Class II H2-IA. b The mice possess point mutations in their alleles. Mouse strains, along with their MHC haplotypes and alloantigens, are listed in Table 1.

[0195] (Table 1) Mouse MHC halotypes and alloantigens TIFF2026518831000002.tif57160

[0196] C57BL / 6J is a wild-type (WT) mouse strain with haplotype H2b (Figure 1A). The MHC haplotype antigen of C57BL / 6J mice is H-2D b H-2K b , and IA b This is B6.C-H2-K bm1 / ByJ(bm1) is the variant allele H2-K bm1 This is an MHC class I mutant possessing H2-K (Figure 1B). bm1 The alleles are H2-K b Seven nucleotides are different, resulting in three amino acid substitutions along the edge of the peptide bond groove at positions 152, 155, and 156 of the α2 domain. B6.C-H2-K bm1 / ByJ(bm1) is a Halotype H-2D b H-2K bm1 , and IA b It has B6(C)-H2-Ab1 bm12 / KhEgJ(bm12) is the variant allele H2-Ab1 bm12 This is an MHC class II mutant possessing (Figure 1C). H2-Ab1 bm12 The allele is H2-IA bThe allele differs by three nucleotides, resulting in three amino acid substitutions along the edge of the peptide bond groove at positions 67, 70, and 71 of the β1 domain. B6(C)-H2-Ab1 bm12 / KhEgJ(bm12) is a Halotype H-2D b H-2K b , and IA bm12 It has.

[0197] Antigen-presenting cells (APCs, e.g., BMDCs) derived from C57BL / 6J WT mice are wild-type MHC alloantigen H2-D b H2-K b , and H2-IA b It includes. When such an APC is pulsed with the E7 peptide derived from the human papillomavirus (HPV) protein E7, it produces the MHC class I alloantigen H2-D b Only presents the E7 peptide (Figure 2A). B6.C-H2-K bm1 APCs derived from / ByJ(bm1) mice present E7 peptides and are wild-type H2-D b , and wild-type H2-IA b , as well as mutant H2-K which promotes allogeneic support of MHC class I. b Includes (Figure 2B). B6(C)-H2-Ab1 bm12 APCs derived from / KhEgJ(bm12) mice present E7 peptides and are wild-type H2-D b , and wild-type H2-K b , as well as mutant H2-IA which promotes allogeneic support of MHC class II. b This includes (Figure 2C). Figure 2D shows the locus of the mouse H-2 complex, which is also known as the mouse major histocompatibility complex (MHC).

[0198] Syngeneic BMDCs are derived from a single mouse strain (e.g., C57BL / 6J WT) and contain antigenic peptides (e.g., H-2D b Restrictive E7 43-77BMDCs are pulsed with a peptide and injected back into the same mouse strain (e.g., C57BL / 6J WT). Semi-allogeneic BMDCs are taken from one mouse strain (e.g., C57BL / 6J WT), pulsed with an antigen peptide, and injected back into a different mouse strain (e.g., B6.C-H2-K) having at least one different MHC alloantigen. bm1 / ByJ(bm1) or B6(C)-H2-Ab1 bm12 It can be a BMDC injected into / KhEgJ(bm12)). In some embodiments, the semi-allogeneic BMDC is B6.C-H2-K bm1 / ByJ(bm1) Mouse or B6(C)-H2-Ab1 bm12 / KhEgJ(bm12) mice are used to extract antigen peptides (e.g., H-2D b Restrictive E7 43-77 It can be BMDC, which is pulsed with a peptide and injected into C57BL / 6J WT mice.

[0199] Example 2: Production of bone marrow-derived dendritic cells (BMDCs) This example describes a method for generating dendritic cells (DCs) from mouse bone marrow (BM) progenitor cells.

[0200] Dendritic cells (DCs) were generated from bone marrow (BM) progenitor cells using a slightly modified protocol from Lutz et al., J. Immunol. Meth. 223: 77-92 (1999). Simply put, all BM cells were generated from 8-20 week old mice (C57BL / 6J, B6.C-H2-K). bm1 / ByJ(bm1), or B6(C)-H2-Ab1 bm12These were obtained from the femurs and tibias of / KhEgJ(bm12)). They were obtained after washing with a 3 ml syringe with a 25 G needle in cell culture medium containing RPMI-1640 (Gibco, USA) replaced 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 acids (Gibco, USA), 1 mM sodium pyruvate (Gibco, USA), and 50 μM 2-mercaptoethanol (Gibco, USA). These BM cells were centrifuged at 1500 rpm for 5 minutes, and then the erythrocytes were lysed in ammonium chloride-potassium lysis buffer (Lonza, USA). The cells were filtered through a 40 μm filter and washed twice with cell culture medium by centrifugation. On day 0, 2 × 10 6 The cells were seeded into 10 ml of culture medium containing 20 ng / ml recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTeck / Tebu, Frankfurt, Germany) in a 100 mm bacterial petri dish. On day 3, an additional 10 ml of culture medium containing 20 ng / ml GM-CSF was added. On days 6, 8, and 10, half of the supernatant containing the cells was aspirated, washed, resuspended in 10 ml of fresh culture medium containing 20 ng / ml GM-CSF, and returned to the plate. On days 7, 8, 9, 10, and 12, portions of both suspension and loosely attached cell samples were collected and incubated overnight in or without 100 ng / ml lipopolysaccharide (LPS, Sigma-aldrich, Heidelberg, Germany) to examine their growth and maturation. All samples (both mature and immature cells) were measured for the expression of MHC class II (IAIE), CD11c, CD40, CD80, and CD86. Flow cytometry results were used for CD11c. + IAIE +The cell population exceeded 80% after 8 days of culture and reached over 90% after 10 days of culture with GM-CSF (Figure 3A). Mature BMDCs showed elevated expression levels of MHC class II (IAIE), CD40, CD80, and CD86 (Figures 3B-E), suggesting that the majority of mature BMDCs can be used as DC vaccines in vivo.

[0201] Example 3: Preparation of mature BMDCs pulsed at E7 This example demonstrates a method for stimulating BMDCs using antigenic peptides to mature them.

[0202] After culturing for 7-10 days in a medium containing 20 ng / ml GM-CSF, C57BL / 6J(WT), B6.C-H2-K bm1 / ByJ(bm1), or B6(C)-H2-Ab1 bm12 BMDCs derived from / KhEgJ(bm12) were collected and placed in fresh culture medium in batches of 1-2 × 10⁶. 6 Resuspend cells / ml and use E7, a peptide derived from the E7 protein, for antigen pulse treatment. 43-77 Incubated with (10 μg / ml, United Biosystems #241093) for 1-2 hours. E7 43-77 The amino acid sequence is, The filename is TIFF2026518831000003.tif4128. The E7 peptide is H-2D on BMDC. b They bind to MHC molecules (Figure 2). These BMDCs, pulsed with antigens, function as antigen-presenting cells and undergo H-2D b -The E7 peptide complex presents antigens to CD8+ cells, thereby stimulating CD8+ cells to attack TC-1 cancer cells expressing E7, ultimately slowing or stopping cancer growth.

[0203] After pulse processing, C57BL / 6J(WT), B6.C-H2-K bm1 / ByJ(bm1), or B6(C)-H2-Ab1 bm12BMDCs derived from / KhEgJ(bm12) were matured overnight with 100 ng / ml LPS. After overnight incubation, the cells were detached from the petri dish with 2 mM EDTA, washed with PBS to remove peptides and LPS, and resuspended in PBS for further use. CD11c expression in BMDCs was investigated by flow cytometry. After culturing BMDCs from all three mouse strains (WT, bm1, and bm12) with GM-CSF for 10 days, over 90% were CD11c+ (Figure 4A), indicating that the majority of BMDCs are converted to bonefydenocytes (H2D). b (B) The expression of MHC class II (IA / IE), CD40, CD80, and CD86 (C) was also investigated by flow cytometry (Figures 4B and 4C) to confirm the maturation of BMDCs from all three mouse strains (WT, bm1, and bm12).

[0204] Example 4: Mouse tumor model This example demonstrates that MHC class II mutant dendritic cell vaccines pulsed with peptides exhibit superior efficacy in a mouse tumor model.

[0205] Autologous dendritic cell (DC) vaccines containing tumor antigens are used clinically, but their therapeutic effects are limited. Immunotherapy using hemi-allogeneic DCs, while still controversial, could be an alternative source and potentially more attractive than autologous DC vaccines. This is because "off-the-shelf" DCs can be used for a large number of patients without requiring very long individual manufacturing times and can provide additional "allogeneic support." Two mouse studies were conducted to compare the efficacy of syngeneic DC vaccines and hemi-allogeneic DC vaccines and to determine whether therapeutic hemi-allogeneic DC vaccines are more effective in suppressing tumors. Female C57BL / 6 mice were subcutaneously inoculated with TC-1 cells expressing human papillomavirus E6 and E7. Syngeneic bone marrow dendritic cells (BMDCs) were generated from C57BL / 6, and hemi-allogeneic BMDCs expressed MHC class I H2-K bAlleles or MHC class II H2-IA b Two mouse strains each having point mutations limited to alleles, namely B6.C-H2-K bm1 / ByJ and B6(C)-H2-Ab1 bm12 / KhEgJ were generated. Each BMDC was pulsed with H-2D b restricted E7 43-77 peptide and matured before injection. Mice received intradermal injection of one of the syngeneic BMDC vaccines or allogeneic BMDC vaccines pulsed with E7 from 8 to 9 days after TC-1 transplantation. Compared with saline control, MHC class I mutant BMDC vaccine had the same efficacy as syngeneic BMDC vaccine in suppressing TC-1 tumor growth. However, MHC class II mutant BMDC vaccine had significantly better efficacy than other BMDC vaccines. Therefore, MHC class II allogeneic BMDC may be more effective than syngeneic DC-based cancer vaccines, probably because class II alloantigens induce additional T cell help for anti-tumor immunity.

[0206] In the first mouse tumor model study, female C57BL / 6J mice (13 weeks old) were subcutaneously inoculated with TC-1 cells, and then vaccinated intradermally 5 times with either syngeneic E7-pulsed BMDC vaccine or allogeneic E7-pulsed BMDC vaccine. TC-1 cells are a mouse lung cancer cell line that expresses E6 and E7, which are human papillomavirus (HPV) proteins. On the 8th day after inoculation of TC-1 cells, the tumor growth of all mice reached a diameter of about 5 mm. On the 8th, 13th, 19th, 23rd, and 28th days after TC-1 cell inoculation, syngeneic E7-pulsed BMDC (E7-mBMDC WT) derived from C57BL / 6J, or B6.C-H2-K bm1 allogeneic E7-pulsed BMDC (E7-mBMDC bm1) derived from / ByJ, or B6(C)-H2-Ab1 bm12 allogeneic E7-pulsed BMDC (E7-mBMDC bm12) derived from / KhEgJ were 2×10 6 cells (1.6×10 on the 19th day 6(number) were vaccinated intradermally in the skin of the mice. The control group was injected with phosphate-buffered saline (PBS). As shown in Fig. 5A, compared with the PBS control group, vaccination with E7-pulsed BMDC WT resulted in slower tumor growth. E7-pulsed BMDC of bm1 (MHC class I variant) showed the same effect as E7-pulsed BMDC (WT), while E7-pulsed BMDC of bm12 (MHC class II variant) showed a greater effect than either E7-pulsed BMDC (WT) or E7-pulsed BMDC of bm1 (MHC class I variant). The tumor of one mouse injected with E7-pulsed BMDC of bm12 (MHC class II variant) disappeared after the mouse received the fifth vaccination (Fig. 5B).

[0207] More specific tumor measurements (tumor volume increase and tumor weight) of mice after TC-1 cell inoculation and E7-pulsed mBMDC vaccination are shown in Fig. 6. From the tumor volumes on days 19, 21, and 34 after TC-1 cell inoculation, it is shown that all vaccinated mice had slower tumor growth compared to PBS control mice, and that mice vaccinated with E7-pulsed mBMDC of bm12 (MHC class II variant) showed the best response compared to all other groups after the third vaccination on day 19 (Figs. 6A - C). All mice were euthanized, and the tumor weights were measured on day 35. Mice vaccinated with E7-pulsed mBMDC of bm12 (MHC class II variant) had significantly lighter tumors compared to the other three groups (Fig. 6D).

[0208] In a second mouse tumor model study, female C57BL / 6J mice (9 weeks old) were subcutaneously inoculated with TC-1 cells, and then either syngeneic E7-pulsed BMDC vaccine or allogeneic E7-pulsed BMDC vaccine was vaccinated intradermally 4 times. On the 9th day after inoculation, the tumor growth of all mice reached a diameter of approximately 5 mm. On days 9, 14, 19, and 24 after TC-1 cell inoculation, syngeneic E7-pulsed BMDC (E7-mBMDC WT) derived from C57BL / 6J, or B6.C-H2-K bm1 / ByJ-derived semi-isolated E7 pulse BMDC (E7-mBMDC bm1), or B6(C)-H2-Ab1 bm12 / KhEgJ-derived semi-isolated E7 pulse BMDC (E7-mBMDC bm12) 2 × 10 6 (1.6 x 10 on day 14) 6 The mice were vaccinated intradermally with E7-BMDC WT cells. The control group was injected with phosphate-buffered saline (PBS). Similar to the first mouse tumor model study, tumor growth was slower in mice vaccinated with E7-BMDC WT compared to the PBS control group. Mice vaccinated with E7-BMDC bm1 (MHC class I mutant) showed a similar effect to mice vaccinated with E7-BMDC WT. Mice vaccinated with E7-BMDC bm12 (MHC class II mutant) showed a greater effect than either E7-BMDC WT or E7-BMDC bm1 (MHC class I mutant), as demonstrated by the tumor growth in the mice (Figure 7A). On day 28 after TC-1 cell inoculation, a significant difference in tumor / body weight ratio was observed between the PBS control group and the E7-pulsed BMDC group with bm12 (MHC class II mutant) or the E7-pulsed BMDC group with bm1 (MHC class I mutant) (Figure 7B). The tumor / body weight ratio in the bm12 (MHC class II mutant) E7 pulsed BMDC group was also significantly lower than in the E7 pulsed BMDC (WT) group (Figure 7B). Measurements of mouse body weight (Figure 7C) and tumor weight (Figure 7D) for mice at day 28 also showed that mice vaccinated with bm12 (MHC class II mutant) E7 pulsed mBMDC showed a greater effect than the other three groups.

[0209] Conclusion: Treatment with a semi-allogeneic cancer antigen pulsed BMDC containing bm12 (MHC class II mutant) was more potent in limiting tumor growth than with a syngeneic cancer antigen pulsed BMDC. This suggests that the allo-CD4+TH response induced by the bm12 molecule additionally assists the BMDC, enhancing its antigen-presenting ability and resulting in a more potent anti-cancer response.

[0210] Example 5: Mouse tumor model with CD4 T cell depletion and / or CD8 T cell depletion This example demonstrates that the removal of CD4 T cells assists a hemi-allogeneic cancer vaccine in limiting late-stage tumor growth in a mouse tumor model.

[0211] Female C57BL / 6J mice (10 weeks old) 5 TC-1 cells were subcutaneously inoculated. On day 8 after inoculation, all mouse tumors reached a diameter of approximately 5 mm. Intraperitoneal injection of anti-CD4 antibody (early aCD4) or anti-CD8 antibody (aCD8) was started on day 6 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. Intraperitoneal injection of anti-CD4 antibody (late aCD4) was started on day 17 after inoculation (200 μg / mouse) and continued every 2-4 days (100 μg / mouse) until the end of the experiment. On days 7, 12, 17, 22, and 27 after TC-1 cell inoculation, syngeneic E7 pulsed BMDC (E7-mBMDC WT) or B6(C)-H2-Ab1 derived from C57BL / 6J was administered. bm12 / KhEgJ-derived semi-isolated E7 pulse BMDC (E7-mBMDC bm12) 2 × 10 6 The mice were vaccinated intradermally. The control group was injected with phosphate-buffered saline (PBS).

[0212] As shown in Figure 8A, compared to the PBS control group, vaccination with E7-BMDC WT and isotype control antibody (isotype + E7-BMDC WT) slowed tumor growth. However, E7-BMDC bm12 (MHC class II mutant) and isotype control antibody (isotype + E7-BMDC bm12) showed a greater tumor growth restriction effect than E7-BMDC WT and isotype control antibody (isotype + E7-BMDC WT). Mice vaccinated with E7-BMDC WT and isotype control antibody (isotype + E7-BMDC WT), and mice vaccinated with E7-BMDC WT and treated with anti-CD4 antibody in the early stages of tumor growth (early aCD4 + E7-BMDC WT) showed similar tumor growth curves (Figure 8B). Mice vaccinated with E7-BMDC bm12 and isotype control antibody (isotype + E7-mBMDC bm12) showed a greater tumor growth restriction effect (Figure 8C). When anti-CD4 antibodies were administered to vaccinated mice in the early stages of tumor growth (early aCD4+ E7-mBMDC bm12), such effects were suppressed until day 17. However, as further shown in Figure 8C, when anti-CD4 antibodies were administered to vaccinated mice in the later stages of tumor growth (late aCD4+ E7-mBMDC bm12), the highest tumor growth suppression effect was observed after the fifth vaccination. This indicates that CD4 removal can contribute to E7-BMDC bm12's ability to fight tumors in the later stages of tumor growth.

[0213] Figure 9 shows the increase in tumor volume in mice 14 days (Figure 9A), 20 days (Figure 9B), and 29 days (Figure 9C) after TC-1 cell inoculation in studies involving the removal of CD4 T cells or CD8 T cells. As shown in Figure 9A, in the early stages of tumor growth, the tumor growth-limiting effect of E7-BMDC bm12 was significantly reduced by the removal of CD4 T cells, indicating that the allo-CD4+Th response induced by the semi-allogeneic cancer antigen pulsed BMDC bm12 (MHC class II mutant) is very important for controlling tumor growth in the early stages. However, as shown in Figure 9C, CD4 + CD25+ In the late stages of tumor growth, when suppressor T cells begin to be upregulated in the immune system, the removal of CD4 T cells assisted the restriction of tumor growth by the E7-mBMDC bm12 vaccine. These data suggest that CD4 T cells in the late stages of tumor growth + CD25 + The removal of CD4 T cells, most likely through the removal of suppressor T cells, is shown to contribute to limiting tumor growth with hemi-allogeneic cancer vaccines.

[0214] Example 6: Mouse tumor model with Treg cell removal This embodiment demonstrates that Treg removal assists a semi-allogeneic cancer vaccine in limiting tumor growth in a mouse tumor model.

[0215] CD4 + CD25 + To further investigate whether the removal of suppressor T cells or regulatory T cells (Treg cells) can contribute to the suppression of mouse tumor growth by E7-mBMDC bm12, six groups of female B6.129(Cg)-Foxp3tm3(Hbegf / GFP)Ayr / J mice (10 weeks old) were subjected to 1 × 10⁶ doses. 5 TC-1 cells were subcutaneously inoculated. On day 7 after inoculation, all mouse tumors reached a diameter of approximately 5 mm. The first group of mice was injected with phosphate-buffered saline (PBS) only as a control group. The second group of mice (two E7-mBMDC bm12) were injected with B6(C)-H2-Ab1 on days 3 and 13 after TC-1 cell inoculation. bm12 / KhEgJ-derived semi-isolated E7 pulse BMDC (E7-mBMDC bm12) 2 × 10 6 Each mouse was vaccinated intradermally. Three groups of mice (E7-mBMDC bm12, 5 doses) received the same vaccine on days 8, 13, 18, 23, and 28. These Foxp3 DTRKnock-in mice express the human diphtheria toxin (DT) receptor, and Treg cells decrease upon injection of DT. Of the two "two doses of E7-mBMDC bm12" groups, one group (late DT + two doses of E7-mBMDC bm12) received late DT injections, starting with an intraperitoneal injection of 10 μg / kg DT 15 days after inoculation, while the other group received no DT treatment (two doses of E7-mBMDC bm12). Of the three "5 doses of E7-mBMDC bm12" groups, one mouse group (initial DT + 5 doses of E7-mBMDC bm12) received an initial DT injection of 10 μg / kg DT intraperitoneally starting on day 6 after inoculation, another mouse group (late DT + 5 doses of E7-mBMDC bm12) received a late DT injection starting on day 15 after inoculation, and the last group received no DT treatment (5 doses of E7-mBMDC bm12). All mice that received DT treatment continued to receive DT treatment every 2-4 days until the end of the experiment.

[0216] As shown in Figure 10, compared to vaccination with E7-BMDC bm12 (MHC class II mutant) alone, vaccination with E7-BMDC bm12 combined with early DT-mediated Treg cell removal resulted in better delayed tumor growth suppression, but only in the early stages of tumor growth. However, when two or five doses of E7-BMDC bm12 vaccination were combined with late DT-mediated Treg cell removal, stronger delayed tumor growth suppression was observed, but late Treg removal with five doses of E7-mBMDC bm12 showed a greater effect than late Treg removal with two doses of E7-mBMDC bm12.

[0217] Compared to mice vaccinated with E7-BMDC bm12 alone (either 2 or 5 doses of E7-mBMDC bm12), DT-treated vaccinated mice showed reduced tumor growth. Specifically, treatment with "early DT + 5 doses of E7-BMDC bm12" slowed tumor growth in the early stages; treatment with "late DT + 2 doses of E7-BMDC bm12" slowed tumor growth in the later stages; and treatment with "late DT + 5 doses of E7-BMDC bm12" showed the greatest effect in controlling tumor growth in the terminal stages, and also demonstrated tumor suppression at the end of the experiment. These data suggest that Treg cell removal assists in limiting tumor growth in mice with hemi-allogeneic cancer vaccines.

[0218] Example 7: IFNγ production in TC-1-containing T cells co-cultured with BMDC vaccine This example describes IFNγ production in TC-1-containing mouse T cells co-cultured with mBMDC WT, E7-mBMDC WT, mBMDC bm12, and E7-mBMDC bm12.

[0219] CD4 T cells and CD8 T cells were isolated from the spleen of TC-1-containing mice 8 days after TC-1 inoculation and stored in a 96-well round plate, with 2 × 10 cells per well. 5 Incubate at individual concentrations for 24 or 72 hours, either alone or in BMDC (1 × 10 cells per well). 5The cells were co-cultured with (or untreated with E7 pulse treatment). Isolated CD8 T cells were examined either alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDC WT (E7-WT CD8), mBMDC bm12 (bm12 CD8), or E7-pulsed mBMDC bm12 (E7-bm12 CD8). IFNγ production was investigated by intracellular staining and flow cytometry (Figure 11A), and IFNγ production in the supernatant was examined by ELISA (Figure 11C). Isolated CD4 T cells were cultured either alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDC WT (E7-WT CD4), mBMDC bm12 (bm12 CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD4). IFNγ production was examined by intracellular staining and flow cytometry (Figure 11B), and IFNγ production in the supernatant was examined by ELISA (Figure 11C). 2×10 5 individual CD8 T cells and 2 × 10⁶ 5 A mixture of individual CD4 T cells was examined similarly, either alone (CD8+CD4) or co-cultured with mBMDC WT (WT CD8+CD4), E7-pulsed mBMDC WT (E7-WT CD8+CD4), mBMDC bm12 (bm12 CD8+CD4), or E7-pulsed mBMDC bm12 (E7-bm12 CD8+CD4) (Figures 11A-C).

[0220] As shown in Figure 11A, CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8) produced significantly higher levels of IFNγ compared to a mixture of CD8 T cells 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 mixture of CD8 T cells and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4) showed similar high levels of IFNγ production as CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8). These data indicate that the allo CD4+ Th response induced by E7-mBMDC bm12 vaccination can stimulate CD8 cells to produce high levels of IFNγ. As shown in Figure 11B, CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) produced significantly higher levels of IFNγ compared to CD4 T cells co-cultured with E7-mBMDC WT (E7-WT CD4). Figure 11C shows IFNγ production in the supernatant obtained from cultures of CD4 T cells and / or CD8 T cells examined by ELISA 72 hours after co-culture. Although IFNγ was not detected in most samples, CD4 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD4) showed a significantly greater amount of IFNγ release than CD8 T cells co-cultured with E7-mBMDC bm12 (E7-BM12 CD8). Robust IFNγ production was observed in the supernatant of a mixture of CD8 T cells and CD4 T cells co-cultured with E7-mBMDC bm12 (E7-bm12 CD8+CD4), indicating that co-culturing CD8 T cells and CD4 T cells has a synergistic effect on IFNγ production.

[0221] V References 1. Lutz MB, Kukutsch N, Ogilvie AL, Rossner 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.

[0222] The foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, and those skilled in the art will appreciate that some changes and modifications may be practiced within the scope of the appended claims. Further, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference.

[0223] Unofficial Array List E7 43-77 : TIFF2026518831000004.tif4128

Claims

1. Engineered mammalian dendritic cells containing one or more exogenous alleles for at least one major histocompatibility complex (MHC) class II gene.

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

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

4. The engineered mammalian dendritic cell according to claim 1 or 2, wherein the exogenous allele comprises a first exogenous allele for an MHC class II gene and a second exogenous allele for the same MHC class II gene.

5. The manipulated mammalian dendritic cell according to any one of claims 1 to 4, which is a manipulated human dendritic cell.

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

7. The manipulated mammalian dendritic cell according to 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 manipulated mammalian dendritic cell according to claim 7, wherein the HLA-DR gene comprises the HLA-DRA gene, the HLA-DRB1 gene, the HLA-DRB3 gene, the HLA-DRB4 gene, the HLA-DRB5 gene, or a combination thereof.

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

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

11. A manipulated mammalian dendritic cell according to any one of claims 1 to 10, comprising a pathogen antigen, a tumor-associated antigen, a neoantigen, an allergen, an antigen targeted by an autoimmune response, or a fragment thereof.

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

13. The manipulated mammalian dendritic cell according to claim 12, wherein the cell line is HL-60, THP-1, K562, MUTZ3, or immortalized dendritic cell.

14. The engineered mammalian dendritic cells according to claim 13, wherein the immortalized dendritic cells express HTLV-1 transactivator (Tax) protein, SV40 protein, and / or hTERT.

15. The manipulated mammalian dendritic cell according to any one of claims 1 to 11, wherein the cell is artificially produced from primary cells.

16. The manipulated mammalian dendritic cells according to claim 15, wherein the primary cells are derived from a patient.

17. The manipulated mammalian dendritic cells according to claim 16, wherein the patient has cancer.

18. A composition comprising manipulated mammalian dendritic cells according to any one of claims 1 to 17.

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

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

21. A method for immunotherapy using hemi-allogeneic dendritic cells in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 19 or 20 to the subject.

22. Before the administration step, (i) A step of obtaining an MHC class II allele profile by determining the genotype of multiple MHC class II genes in a biological sample derived from the subject; and (ii) A step of selecting engineered mammalian dendritic cells for administration to the subject, wherein the engineered mammalian dendritic cells contain one or more mismatches with respect to the subject's MHC class II allele profile. The method according to claim 21, further comprising:

23. The method according to claim 21 or 22, further comprising the step of administering a regulatory T cell inhibitor (Treg activator) to the subject.

24. The method according to claim 23, wherein the Treg-activating substance is selected from the group consisting of antibodies, small molecules, antibody-drug conjugates, immunotoxins, peptide-drug conjugates, peptides, small interfering RNA (siRNA), siRNA conjugates, chemotherapeutic agents, and any derivatives, fragments, or fusions thereof.

25. The method according to claim 23 or 24, wherein the Treg-activating substance is administered after the administration of the pharmaceutical composition.

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

27. The method according to claim 26, wherein the human has cancer, and the manipulated dendritic cells contain a tumor-specific antigen or a fragment thereof.

28. A method for immunotherapy using autologous dendritic cells in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 19 or 20, wherein the manipulated mammalian dendritic cells are derived from the subject's primary immune cells.

29. Before the administration step, (i) A step of obtaining the primary immune cells or a plurality of such primary immune cells from the subject; (ii) The step of determining the genotype of multiple MHC class II genes in the primary immune cell in order to determine the endogenous MHC class II allele profile; and (iii) Below: (a) Introducing one or more exogenous MHC class II alleles containing at least one mismatch with the endogenous MHC class II allele profile of the subject into the primary immune cells; and (b) Introducing pathogen antigens, tumor-associated antigens, neoantigens, allergens, antigens targeted by autoimmune responses, or fragments thereof into primary immune cells. The process of manipulating primary immune cells into mammalian dendritic cells. The method according to claim 28, further comprising:

30. (iii) (c) Incubating the primary immune cells with lipopolysaccharide (LPS), interferon-γ (IFN-γ), or a combination thereof. The method according to claim 29, further comprising:

31. The method according to any one of claims 28 to 30, further comprising the step of administering a Treg-activating substance to the subject.

32. The method according to claim 31, wherein the Treg-activating substance is selected from the group consisting of antibodies, small molecules, antibody-drug conjugates, immunotoxins, peptide-drug conjugates, peptides, small interfering RNA (siRNA), siRNA conjugates, chemotherapeutic agents, and any derivatives, fragments, or fusions thereof.

33. The method according to claim 31 or 32, wherein the Treg-activating substance is administered after the administration of the pharmaceutical composition.

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