WO2026003069A1

WO2026003069A1 - Constructs and vectors for reprogramming cells to cdc1 cells, compositions and methods thereof

Info

Publication number: WO2026003069A1
Application number: PCT/EP2025/067899
Authority: WO
Inventors: Fábio Alexandre Fiúza ROSA; Cristiana Ferreira Pires; Carlos Filipe Ribeiro Lemos Pereira; Ervin ASCIC
Original assignee: Asgard Therapeutics AB
Current assignee: Asgard Therapeutics AB
Priority date: 2024-06-25
Filing date: 2025-06-25
Publication date: 2026-01-02
Anticipated expiration: 2026-12-25
Also published as: WO2026003067A1

Abstract

The present disclosure relates to the optimization of direct cell reprogramming tools and methods, in particular for cDC1 cell reprogramming. The present invention provides constructs and methods enhancing cDC1 reprogramming, in particular in vivo, cells obtained thereof, and medical uses thereof.

Description

Constructs and vectors for reprogramming cells to cDC1 cells, compositions and methods thereof

Technical field

The present invention relates to constructs, vectors and methods for reprogramming cells into dendritic or antigen-presenting cells, and uses thereof. In particular, the present invention relates to constructs and adenoviral vectors enabling reprogramming of cells into dendritic or antigen-presenting cells with enhanced reprogramming efficiency.

Background

Cancer immunotherapies rely on the establishment of tumor antigen-specific T cell responses. T cells identify tumor antigens displayed on tumor cells' major histocompatibility complexes (MHC) and carry out their functions by killing cells and producing inflammatory cytokines. However, tumor cells often fail to activate T cells due to downregulation of antigen presentation pathways, the creation of an immunosuppressive tumor microenvironment (TME), and the absence or dysfunction of professional antigen presenting cells, such as dendritic cells (DCs). This presents a challenge for achieving widespread success with current cancer immunotherapy approaches. For example, immune checkpoint blockade (ICB), while transforming solid tumor treatment, yields a response rate of only 60% in melanoma patients treated with anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T lymphocyte- associated protein 4 (CTLA-4). Other less immunogenic cancer types, like breast cancer, microsatellite-stable colorectal cancer, and glioblastoma, exhibit resistance to immunotherapy, with long-term immunity induced in less than 5% of patients.

Growing evidence suggests that conventional dendritic cells type 1 (cDC1s) play a crucial role in T cell-mediated tumor regression and response to ICB across various cancer types. cDC1s, a rare subset of DCs, express high levels of MHC class I and II, the co-stimulatory molecule CD40, and specific markers like XCR1 and CLEC9A (Cabeza-Cabrerizo et al. 2021). Within tumors, cDC1s are essential for recruiting and activating T cells through chemokine secretion and antigen cross-presentation, facilitating effective cancer immunity. However, these unique functional attributes of cDC1s have not yet been fully harnessed for immunotherapy due to the lack of methods enabling the generation of a pure population of cDC1s. Cellular reprogramming offers a strategy for generating specific cell types in vivo by overexpression of cell type-specific transcription factors (TFs). In vivo cell fate reprogramming allows the conversion of endogenous somatic cells into different cell types within the organism, offering therapeutic potential directly at the site of disease bypassing the challenges associated with ex vivo cell manufacturing for personalized cell therapies. For example, mouse pancreatic exocrine cells were converted in situ to insulin-secreting p-cells by delivering three transcription factors to the pancreas using adenoviral vectors. Similarly, scar-forming cardiac fibroblasts were transformed into cardiomyocytes in mouse models of myocardial infarction, leading to improved heart function (Qian et al. 2012). Glial cells were converted to functional neurons after brain injury or in models of neurodegenerative diseases (Torper et al. 2015) and rod photoreceptors were generated within the retina, resulting in improved vision (Yao et al. 2018). However, in vivo reprogramming may differ from in vitro conversion processes and the optimization of constructs and methods for in vivo applications is not straightforward. Insulin-producing p-cells and cardiomyocytes displayed enhanced functional properties when generated in vivo, likely due to biochemical and mechanical signals present in the environment (Zhou et al. 2008, Qian et al. 2012). Additionally, the transcription factors Ngn2, Dlx2, or NeuroDI were differentially utilized to induce astrocyte-to-neuron conversion in vitro and in vivo (Guo et al. 2014). Differences in the transcription factor combination requirement and the maturity of the cells reported in these studies demonstrated that the in vivo environment has a significant impact on the reprogramming process, highlighting the need to characterize in vivo reprogramming mechanisms and induced phenotypes.

Previously, a transcription factor combination — PU.1 , IRF8, and BATF3 (PIB) — was identified as sufficient to reprogram fibroblasts or tumor cells into cDC1-like cells in vitro, equipped with the essential signals for T cell activation, antigen presentation, costimulatory molecule expression, and chemokine/cytokine secretion (Rosa et al. 2018, Rosa et al. 2022, Zimmermannova et al. 2023). However, whether cDC1 reprogramming progresses in vivo and can be induced by in situ delivery of reprogramming factors, and whether in situ cDC1 reprogramming of solid tumours allows induction of antitumor immunity is still unknown. A fortiori constructs and methods enabling and/or optimizing said cDC1 reprogramming in vivo are still in demand. Therefore, the optimization of direct cell reprogramming tools and methods remains a challenge, and further improvements are needed, for example to bring the technology to clinical use. Constructs and methods enhancing cDC1 reprogramming, a fortiori in vivo, and medical uses thereof, are still in demand.

Summary

The present invention provides solutions to the above-mentioned challenges and needs.

In one aspect, the invention relates to one or more constructs, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1 , IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late).

A second aspect of the present invention relates to one or more vectors comprising the one or more constructs of the first aspect.

A third aspect of the present invention provides a method of manufacturing the vectors of the second aspect, said method comprising: a) Providing a host cell capable of being transfected with a nucleic acid sequence encoding the vectors, such as the adenoviral vectors described herein, for example the vectors of the second or seventeenth aspect of the present disclosure; b) Transfecting the host cell with the nucleic acid sequence encoding said vectors; c) Culturing the transfected host cell under conditions suitable for expression and assembly of the vectors, such as adenoviral particles; d) Harvesting the vectors from the cultured host cells; and e) Purifying the harvested vectors.

A fourth aspect of the present invention relates to a cell comprising the one or more constructs or the one or more vectors of the first or second aspect, respectively.

A fifth aspect of the present invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, comprising the following step: a) transducing a cell with the one or more constructs or the one or more vectors of the present disclosure.

A sixth aspect of the present invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, comprising the following steps: a) transducing a cell with the one or more constructs or the one or more vectors of the present disclosure; b) expressing the transcription factors whereby a reprogrammed or induced cell is obtained.

A seventh aspect of the present invention relates to a reprogrammed or induced cell obtained by the method of the present disclosure.

An eight aspect of the present invention relates to a pharmaceutical composition comprising the one or more constructs of the first aspect, the one or more vectors of the second aspect, the cell of the third aspect, or the reprogrammed or induced cell of the sixth aspect, and a pharmaceutically acceptable carrier, diluent, or excipient.

A ninth aspect of the present invention relates to the one or more constructs of the first aspect, the one or more vectors of the second aspect, the cell of the third aspect, the reprogrammed or induced cell of the sixth aspect, and/or the pharmaceutical composition of the seventh aspect, for use in medicine.

A tenth aspect of the present invention relates to the one or more constructs of the first aspect, the one or more vectors of the second aspect, the cell of the third aspect, the reprogrammed or induced cell of the sixth aspect, and/or the pharmaceutical composition of the seventh aspect, for use in the treatment of cancer, such as solid tumor cancers and/or hematological cancers.

An eleventh aspect of the present invention relates to a method of treating cancer, the method comprising administering to an individual in need thereof the one or more constructs of the first aspect, the one or more vectors of the second aspect, the cell of the third aspect, the reprogrammed or induced cell of the sixth aspect, and/or the pharmaceutical composition of the seventh aspect.

A twelfth aspect of the present invention relates to the use of the one or more constructs of the first aspect, the one or more vectors of the second aspect, the cell of the third aspect, the reprogrammed or induced cell of the sixth aspect, and/or the pharmaceutical composition of the seventh aspect, for the manufacture of a medicament for the treatment of cancer.

A thirteenth aspect of the present invention relates to a method for determining efficacy to treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell as described herein, comprising: determining the levels of tumor infiltrating lymphocyte (TIL) B cell populations, such as lgM⁺ B cells (CD19+ B220+ lgM+), activated B cells (CD19+ B220+ CD95+ GL7+), plasma cells (CD45+ CD19- CD138+), and/or follicular dendritic cells (DCs) (CD45+ CD19- CD23+) in a first a biological sample of an individual having received the treatment; comparing the levels of the TILs with the levels of the TILs in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said TILs in the first sample compared to the second sample indicates efficacious treatment.

A fourteenth aspect of the present invention relates to a method for determining efficacy to treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell as described herein, comprising: determining the levels of tumor-specific IgM, lgG1, and/or IgA antibodies in a first a biological sample of an individual having received the treatment; comparing the levels of the tumor-specific IgM, IgG 1 , and/or IgA antibodies with the levels of the tumor-specific IgM, lgG1 , and/or IgA antibodies in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said tumor-specific IgM, lgG1 , and/or IgA antibodies in the first sample compared to the second sample indicates efficacious treatment.

Description of Drawings

Figure 1. In vivo cDC1 reprogramming elicits potent antitumor immunity. (A) 88% of B16 cells were transduced with lentiviral vectors encoding PIB-eGFP (PIB: PU.1 , IRF8, and BATF3) or PC-eGFP (PC: PU.1 and C/EBPa) in vitro, mixed with 12% of parental B16 cells and a total of 1x10⁵ cells per mouse were injected subcutaneously to induce tumor cell reprogramming in vivo along with tumor establishment. Injection of eGFP-transduced cells mixed with parental cells were included as controls. Additionally, cell mixtures were maintained in vitro to quantify transduced cells at day 3 and reprogramming efficiency until day 9. ICB (anti-PD-1 and anti-CTU\-4) or isotype control (lgG2a and lgG2b) antibodies were administered intraperitoneally at day 7, 10, and 13 post tumor establishment. Tumor growth (upper panel) and survival of mice (bottom panel) are shown (n=10). (B) Flow cytometry analysis and quantification of the number of surface MHC-I molecules per cell and (H) MHC-II expression in B16 cells by mean fluorescence intensity (MFI) after 3 and 9 days of in vitro reprogramming (n=3). (C) Quantification of proliferating CTV^|OWCD44⁺ ovalbumin-specific CD8⁺ T cells (OT-I) after co-culture with CD103⁺bone marrow-derived dendritic cells (BM-DC), eGFP-transduced B16 cells (not expressing ovalbumin), MACS-enriched CD45⁺ and MHC-IT B16-derived cDC1-like cells or MACS-enriched CD45⁺ and CD11b⁺ B16-derived macrophage-like cells (PC). BM-DC, eGFP-transduced or reprogrammed cells were pulsed for 24 hours with full-length ovalbumin protein. To start the co-culture, ovalbumin-containing media was washed away extensively, and fresh media added together with OT-I cells and cocultures were performed for 72 hours (n=4-7). Data in panel B and C are shown as mean ± SD. Survival analysis in panel A was performed by log-rank Mantel-Cox test.

****p<0.0001. Figure 2. In vivo cDC1 reprogramming elicits systemic and durable antitumor immunity. (A) Mice were injected subcutaneously with melanoma cells (B16, YUMM1.7, B2905, BRAF^V600ECOX1/2^K°) after transduction with PIB-eGFP or control eGFP and mixing 1 :1 with parental cells (measured percentages by flow cytometry at day 3 are indicated) to induce tumor cell reprogramming in vivo along with tumor establishment. Anti-PD-1 , anti-CTLA-4 or isotype control antibodies were administered by intraperitoneal injection at days 7, 10 and 13. T umor growth and survival are shown (n=5- 6). B16, YUMM1.7 and B2905 models were established in C57BL/6J and BRAF^V600ECOX1/2^K° model in BATF3^KO mice. (B) Flow cytometry quantification of tumor antigen-specific IFN-y+CD8+ or IFN-y+CD4+ T cells from peripheral blood at day 14. T cells were isolated and re-stimulated in vitro using an antigen-agnostic approach with IFNy-stimulated melanoma cell lines. (C) Survivor mice that remained tumor-free for 100 days were re-challenged with YUMM1.7 cells (upper panel) or BRAF^V600ECOX1/2^K° (bottom panel). Age-matched naive mice were used as controls and tumor growth and survival are shown (n=2-5). (D) Bilateral YUMM1.7 tumor growth after injection of 1:1 mixtures into the treated flank (right) and untransduced cells into the non-treated flank (left), as monotherapy (PIB-eGFP) or in combination with anti-PD-1 or anti-CTLA-4 (n=10). Data in panels B and D are shown as mean ± SD. Survival analyses in panel A and C were performed by log-rank Mantel-Cox test. Comparisons in panels B and D were analyzed using the Mann-Whitney test, ns - non-significant, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001.

Figure 3. In vivo cDC1 reprogramming remodels the tumor microenvironment.

(A) Hematoxylin and eosin (H&E) staining (top) and immunofluorescence (bottom) analysis of paraffin-embedded YUMM1.7 tumors 9 days after subcutaneous implantation of PIB-eGFP- or control eGFP-transduced cells (1 :2 ratio of transduced to parental cells). Tumor sections were stained for eGFP, CD45 (immune cells) and nuclei (Syto 13) (n=3). Arrows indicate TLS-like structures. Scale bars are 500nm. (B) H&E (upper left) and immunofluorescence of a tertiary lymphoid structure (TLS) in PIB- eGFP tumors stained for CD19 (B cells), CD4 (CD4+ T cells), CD8 (CD8+ T cells) and PDPN (podoplanin+ stromal cells). Dashed lines indicate TLS border. Scale bars are 100pm. (C) Flow cytometry quantification of the percentages of tumor-infiltrating CD19+ B cells, CD49b+CD3- NK cells and CD8+ and CD4+ T cells. (D) Quantification of PD-1+CD8+ and PD-1+CD4+ T cells within the tumor. (E) Percentages of CD44+CD62L- effector memory and CD44+CD62L+ central memory CD8+ and CD4+ T cells. (F) Quantification PD-1+CD25+ regulatory CD8+ T cells and CD25+CD4+ Tregs. (G) Mice were subjected to antibody-mediated depletion of CD8+ T cells (aCD8), CD4+ T cells (aCD4), NK cells (aNK1.1) or isotype controls and tumors established with a mixture of transduced YUMM1.7 cells. Tumor growth is shown (n=10). Data in panel C-F are shown as mean ± SD. Comparisons in panel C-F were analysed using the Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001 , ****p<0.0001 .

Figure 4. In vivo reprogrammed melanoma cells expand polyclonal CD4⁺ T cells. (A) Experimental design for 5' single cell RNA-seq with TCR enrichment. YUMM1.7 tumors were established with a 1 :1 mixture of PIB-eGFP or control eGFP-transduced and untransduced cells. Peripheral blood, tumor-draining lymph nodes (tdLN) and tumors were isolated 21 days after tumor establishment and CD45⁺CD3⁺ T cells were FACS-purified before loading on a 10x Chromium. Additional groups received intraperitoneally anti-PD-1 at days 7, 10 and 13 (n=5). (B) Bar plots show the percentages of different CD8⁺ and CD4+ T cell subsets in tumors. CD8⁺ T cell subsets are numbered from 0 to 8, and CD4⁺ T cell subsets are numbered from 0 to 11. (C) CD8⁺ and CD4⁺ T cells isolated from tumors were color-coded by clonotype size into small (between 1 and 5 cells), medium (between 5 and 20 cells), and large (>20 cells) clones. TCR sequences detected in only one single cell were excluded from this analysis. Bar plots show percentages of CD8⁺ T cells in tumors and their clonotype distribution. The numbers of unique clones are indicated within the bars. Comparisons in B were performed using the exact Binomial test. Relevant statistical comparisons between intratumoral T cells for the conditions eGFP vs. PIB-eGFP and eGFP+anti-PD-1 vs. PIB- eGFP+anti-PD-1 are shown. All statistical comparisons can be found in data file S1.

**p<0.01, ****p<0.0001.

Figure 5. In vivo induction of a cDC1 phenotype in human cancer cells. Human cancer cell lines were transduced in vitro with PIB-eGFP, implanted in NSG mice and isolated at days 3, 5, and 9 for phenotypic profiling by flow cytometry. eGFP-transduced cells were used as controls and in vitro reprogrammed cells for comparison. Reprogramming efficiency was evaluated by flow cytometry as the percentage of CD45⁺HLA-DR⁺ cells (completely reprogrammed) and CD45⁺HLA-DR^_ or CD45 HLA- DR⁺ cells (partially reprogrammed) gated in eGFP⁺ transduced cancer cells. (A) Representative flow cytometry plots and (B) quantification of reprogramming kinetics in vitro and in vivo of the glioblastoma cell line T98G (gated in CD44⁺eGFP⁺ cells), and melanoma lines A375 and A2058 cells (gated in CD44⁺MCSP⁺eGFP⁺ cells) (n=3). (C) Quantification of surface XCR1 (left) and CLEC9A (right) markers, and (D) HLA-ABC molecules per cell (left) and expression of CD40 (right) gated in CD44⁺eGFP⁺ cells for T98G and CD44⁺MCSP⁺eGFP⁺ for melanoma A375 and A2058 after 5 days of in vitro or in vivo reprogramming (n=3). Data in panels C-F are shown as mean ± SD.

Figure 6. Reprogramming progresses in spheroids independently of immunosuppressive tumor environment. Cancer cells were transduced with PIB- eGFP or PIB-mCherry and used to form spheroids (3D) or cultured in monolayer (2D). (A) Representative flow cytometry plots showing phenotype of reprogrammed T98G and A375 cells in 2D and 3D compared to eGFP-transduced cells 9 days after transduction. Reprogramming efficiency was evaluated by flow cytometry as the percentage of CD45⁺HLA-DR⁺ cells (completely reprogrammed) and CD45⁺HLA-DR' or CD45'HLA- DR⁺ cells (partially reprogrammed). (B) Reprogrammed and partially reprogrammed T98G cells were purified at reprogramming days 3, 7, and 9 and profiled by scRNA-seq. Heatmap shows the percentage of cells transcriptionally affiliated with individual DC subsets after integration of scRNA-seq data with data from published DC subsets (GSE94820) (Villani et al. 2017). (C) Heatmap showing percentage of tumor-APC gene signature activation (Zimmermannova et al. 2023). (D) Gene set enrichment analysis for immunogenic signature (TLR-induced maturation) and tolerogenic signature (homeostatic maturation) (Ardouin et al. 2016) during reprogramming in 2D and 3D. Color gradient depicts enrichment score. (E) Gene set enrichment analysis for interferon (IFN) signature, KEGG Toll-like Receptor (TLR) signaling, STING and NF-KB signaling pathways in T98G cells at day 9 of 3D reprogramming (3D PIB, d9) compared to parental cell line (dO, eGFP-transduced cells). (F) Effect of immunosuppression in cDC1 reprogramming was evaluated using spheroids containing T98G-eGFP⁺ cells combined with cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs) or pericytes at indicated ratios. Bar plots show flow cytometry quantification of reprogramming efficiency gated in T98G-eGFP⁺ mCherry⁺ cells in spheroids with increasing proportions of CAFs (n=3-9, left), MDSC (n=3. middle), and pericytes (n=6-7, right). CAF07 and MDSC1 refer to cells from one individual donor. (G) T98G cells were transduced with PIB-eGFP or eGFP and maintained either in 2D or used for spheroid formation. 2D and 3D cultures were reprogrammed for 9 days in the presence of increasing concentrations of immunosuppressive cytokines IL-6, TGF-p, VEGF, and immuno-regulatory GM-CSF (indicated in x-axis), and reprogramming efficiency was evaluated at day 9 (n=7-11). Data in panel F and G are shown as mean ± SD. Comparisons in panels F and G were analyzed using two-way ANOVA followed by Tukey's multiple comparison test. *p<0.05; ****p<0.0001.

Figure 7. Adenoviral vectors allow efficient delivery of cDC1 reprogramming factors to mouse and human cancer cells in vitro and in vivo. Lentiviral (LV), adenoviral serotype 5 (Ad), and adeno-associated viral (AAV) transduction and cDC1 reprogramming efficiencies were quantified using mouse and human cancer cell lines and patient-derived cancer cells in monolayer (2D), spheroids (3D), and tumors in vivo. Transduction and reprogramming efficiencies were evaluated by flow cytometry. Lentiviral vectors encoding eGFP (LV-eGFP) were included as control. (A) Flow cytometry quantification of reprogramming efficiency measured by the surface expression of CD45 and M HC-I I, and (B) MHC-I in mouse cancer cell lines at day 3 after transduction (n=2-4). (C) Flow cytometry quantification of reprogramming efficiency measured by the surface expression of CD45 and HLA-DR in human cancer cell lines at day 9 after transduction (n=4). (D) Reprogramming in human melanoma IGR39 measured by cumulative percentages of CD45⁺ and HLA-DR⁺ cells at days 0, 3, 6, 9, and 12 after transduction (n=2). (E) Flow cytometry quantification of reprogramming efficiency in patient-derived cancer cells 9 days post transduction in 2D monolayer or 3D spheroids with PIB-encoding LV, Ad and AAV vectors (LV-PIB-eGFP, adenoviral vector serotype 5 Ad-Pl B-eGFP, AAV-PIB-eGFP) (n=2-3). (F) CD40 and HLA-ABC expression in patient-derived cancer cells 9 days after transduction with LV or Ad vectors serotype 5 in 2D (n=3). (G) Experimental design to evaluate transduction efficiency in situ using subcutaneous B16 tumors in C57BL/6J mice. Tumors were injected with 2 doses of LV- eGFP, adenoviral vector serotype 5 Ad-eGFP, AAV-eGFP vectors or PBS at day 7 and 9 and isolated at day 12 for analysis (n=9-25). (H) Flow cytometry quantification of eGFP⁺ cells of tumors transduced with the 3 viral vectors or PBS. Quantification of viral particles is shown. (I) Human SKLMS1 and A2058 tumors were established in NXG mice and injected 4 times intratumorally with adenoviral vector serotype 5 Ad-Pl B-eGFP or adenoviral vectors serotype 5 Ad-Stuffer-eGFP at day 7, 9, 11 and 13 and analyzed at day 16 by flow cytometry. Flow cytometry quantification of reprogramming efficiency gated in eGFP⁺ cells (n=8-10). Comparisons between CD45⁺HLA-DR⁺ populations (red) were used for statistical analysis. (J) YUMM1.7 tumors were established with decreasing doses of PIB-eGFP-transduced cells mixed with parental cell line. Percentages of reprogrammed cells (CD45⁺ and MHC-II⁺) in cell mixtures were quantified by flow cytometry at day 9 post transduction from parallel in vitro cultures. Tumor growth and survival (bottom) are shown (n=10). The number of complete responses (CR) over the total number of mice per group is indicated. (K) Quantification of CD8⁺ T cell numbers (left), and percentages of effector CCR7'CD45RA'CD8⁺ (middle) and cytotoxic CD95⁺CD8⁺ T cells (right) after 8 days of co-culture with adenoviral vector serotype 5 Ad-eGFP or adenoviral vector serotype 5 Ad-Pl B-eGFP transduced M2778 cells with (50%) or without (100%) CAFs in 2D. (K) Quantification of CD8⁺ T cell numbers within spheroids. Data in panels A to F, H, I, K and L are shown as mean ± SD. Comparisons in panels B were analyzed using One-Way ANOVA followed by Dunn's multiple comparison test. Comparisons in panels I, K and L were analyzed using Mann Whitney test. Survival analysis in panel J was performed by log-rank Mantel-Cox test, ns - nonsignificant; *p<0.05, **p<0.01; ****p<0.0001.

Figure 8. Adenoviral vector-mediated in situ cDC1 reprogramming elicits systemic and long-term antitumor immunity. (A) Experimental design to assess antitumor efficacy of Ad-PIB gene therapy in C57BL/6J mice with subcutaneous B16 melanoma tumors. Tumors were injected 4 times with adenovirus serotype 5 - PIB, Ad-PIB (dark), non-coding Ad vector serotype 5 control (Ad-Stuffer, grey), or PBS (black) at day 7, 9, 11, and 13 after tumor establishment. Anti-PD-1 and anti-CTLA-4 (ICB) were administered intraperitoneally at day 7, 10, and 13. Survivor mice were further subcutaneously re-challenged with B16 cells at day 100 and intravenously at day 160. Grey box indicates the time of treatment. (B) Tumor growth (left) and survival (right) (n=8- 10). The number of complete responses (CR) over the total number of mice per group is indicated. (C) Flow cytometry quantification of turn or- infiltrating CD45+ immune cells and CD8+ T cells at day 16 (n=7-10). (D) Correlation of CD8⁺ T cell infiltration and tumor size. (E) Percentages of intratumoral T-bet⁺PD-T effector, T-bet⁺PD-1⁺ exhausted, and T-bet PD-T terminally exhausted CD8⁺ T cells. Comparisons between the indicated color- coded populations were used for statistical analysis. (F) Ratio of intratumoral T- bet⁺CD44⁺CD4⁺ T helper (Th) cells and CD44⁺CD25⁺ T regulatory (Treg) cells. (G) Flow cytometry quantification of tumor antigen p15E-specific CD8⁺ T cells in tumor-draining lymph nodes (tdLN) and non-draining lymph nodes (n=7-10). (H) Survivor mice and naive control mice were re-challenged subcutaneously with B16 cells. Tumor growth (left) and survival (right) are shown (n=4-5). (I) Flow cytometry quantification of gp100/pmel tumor antigen-specific T cells from peripheral blood at day 14 after in vitro re-stimulation with gp100/pmel peptide. Percentages of p15E-specific IFNg⁺CD44⁺CD62L' effector memory CD8⁺ T cells are shown (n=4-5). (J) Survivor mice were further re-challenged intravenously with B16 cells. Exemplificative images of lungs from survivor and naive mice 14 days after re-challenge (left). Quantification of lung metastatic foci 21 days after intravenous challenge is shown on the right (n=4). (n=4). (K) Mice were subcutaneously inoculated with murine B16 melanoma cells on both flanks. After both tumors emerged, one tumor was treated at a tumor volume of 30-100 mm3 with adenoviral vectors encoding PU.1 , IRFS and BATF3 (Ad5-PIB, n=11) or a noncoding stuffer sequence (Ad5-Stuffer, n=9) at day 11 , 13, 15, 17 post tumor establishment. Mice were also treated with anti-PD1 and anti-CTLA-4 by intraperitoneal administration at day 11, 14 and 17. Tumor growth kinetics of non-treated (left) and treated (right) tumors with Ad5-PIB (upper panel) and Ad5-Stuffer control (bottom panel) vectors. Arrows indicate treatment timings and lines depict individual mice. (L) Analysis and quantification of area under the curve until day 20 of non-treated tumors categorized by Ad5-Stuffer-treated mice, and non-responding (NR) or responding (R) PIB-treated mice. (M) Analysis of survival over time. Data in panels C, E, F, G, I, J and L are shown as mean ± SD. Comparisons in panel C, E, F and J were analyzed using One-Way ANOVA followed by Dunn's multiple comparison test. Comparisons in panels G, I, J and L were analyzed using the Mann Whitney test. Survival analyses in panel B, J and M was performed by log-rank Mantel-Cox test. *p<0.05, **p<0.01; ***p<0.001.

Figure 9. Multiple dosing improves in vivo tumor cell transduction and treatment efficacy. (A) B16 melanoma tumors were treated with 1 , 2 or 4 intra-tumoral injections of Ad5-GFP at day 13; or 13 and 15; or 13, 15, 17 and 19, respectively, after tumor establishment. Tumors treated with 4 intratumoral injections of PBS on days 13, 15, 17 and 19 were included as control. Transduction efficiency was profiled by flow cytometry analysis 9 days after the first intra-tumoral injection as the percentage of GFP+ cells in the live cells isolated from dissociated tumors. CD44+CD45- cancer cells and CD45+ hematopoietic cells at day 9 after the first intratumoral injection (n=6-8). (B) B16 melanoma tumors were treated with 1 , 2 or 4 intra-tumoral injections of Ad5 vectors encoding PU.1 , IRF8 and BATF3 (Ad5-PIB, n=7-8), a non-coding stuffer sequence (Ad5- Stuffer, n=6-7) or PBS (n=5) at day 14, 16, 18 and 20 post tumor establishment. Mice were also treated with anti-PD1 and anti-CTLA-4 (ICB) at days 14, 17 and 20 after tumor establishment. (C) Kinetics of tumor growth after 1 (left), 2 (middle) or 4 (right) intratumoral injections. Arrows correspond to number of injections and grey area to dosing period. (D) Quantification of tumor growth by area under the curve until day 19. (E) Analysis of survival over time. Mean ± SEM is represented. Comparison of survival between groups were performed using Mantel-Cox test and area under curve using Welsh t test in GraphPad Prism 9 software. P values are shown when relevant ^nonsignificant (ns); *p<0.05; p**<0.01).

Figure 10. PIB overexpression mediated by SFFV promoter allows high cDC1 reprogramming efficiency across human cancer cell lines and primary samples.

(A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding for PU.1 , IRF8 and BATF3 under the control of different promoters 3 days after transduction of melanoma A2058, sarcoma SK-LMS-1 , glioblastoma T98G and head and neck Ca9-22 human cancer cell lines using four different multiplicities of infection (MOIs: 0.1x10², 0.5x10³, 1x10³ and 5x10³ 1 FU/cell) and

(B) 9 days after transduction in breast B0845, melanoma M2778 and colorectal CRC DT01950 human primary samples transduced at two different MOIs (0.1x10³ and 1x10³ IFU/cell), measured as percentage of transduced cells expressing CD45 and HLA-DR. SFFV: spleen focus-forming virus promoter, CMV: Human cytomegalovirus immediate early enhancer/promoter, CMV+intron: Human cytomegalovirus immediate early enhancer/promoter fused with the splicing signal from the human beta-globin intron 2, hPGK: Human phosphoglycerate kinase 1 promoter, MNDU3: Promoter of U3 region of the MND retroviral vector, RSV: Rous sarcoma virus long terminal repeat promoter, EF1cc Human eukaryotic translation elongation factor 1 a1 promoter, CAG: CMV early enhancer fused to modified chicken p-actin promoter, CBh: CMV early enhancer fused to modified chicken [3-actin promoter, CBA: CMV early enhancer fused to chicken p-actin promoter and SV40 intron, TERT: Human telomerase reverse transcriptase promoter. The three phenotypes of cDC1 reprogrammed cells are represented: CD45+HLA-DR-, CD45-HLA-DR+ and CD45+HLA-DR+ (n=2). Mean ± SD is shown.

Figure 11. Addition of a WPREmut6 downstream the tricistronic cassete allows higher cDC1 reprogramming efficiency across human cancer cells. (A) Flow cytometry quantification of cDC1 reprogramming efficiency in T98G human cancer cells 3 days after transduction with Ad5 and Ad5/F35 adenoviral vectors encoding PU.1, IRF8 and BATF3 (PIB) in a tricistronic cassete followed by a Woodchuck Hepatitis Virus Post- T anscriptional Regulatory Element (WPRE) sequence. Adenoviral vectors encoding PIB without WPRE or encoding eGFP only were included as controls. Reprogramming efficiency was measured by the frequency of transduced GFP+ cells expressing CD45 and/or HLA-DR (n=2). Mean ± SD are represented. (B) Flow cytometry quantification of CD45 and HLA-DR expression in human cancer cell lines 3 days after transduction with adenoviral vectors serotype 5 encoding for PIB followed by WPRE or the mutated derivative mut6 (mut6WPRE or WPREmut6) at four different MOIs (1x10², 5x10², 1x10³ and 5x10³ IFU/cell). (C) Representative flow cytometry plots showing reprogramming efficiency using a MOI of 1x10³ IFU/cell at day3 (n=2). Mean ± SD are represented.

Figure 12. The Rabbit beta-globin polyadenylation signal (rBGpA) Post- Transcriptional Regulatory Element enhances cDC1 reprogramming efficiency. (A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding for PU.1, IRF8 and BATF3 containing different types of polyadenylation signals 3 days after transduction of melanoma A2058, sarcoma SK- LMS-1, glioblastoma T98G and head and neck Ca9-22 human cancer cell lines using four different MOIs (1x10², 5x10², 1x10³ and 5x10³ IFU/cell) and (B) 9 days after transduction in breast B0845 and melanoma M2778 human primary samples transduced at two different MOIs (1x10² and 1x10³ IFU/cell), measured as percentage of transduced cells expressing CD45 and HLA-DR. (n=2). Mean ± SD are represented. BGH: Bovine Growth Hormone, TK: Herpes Simplex Virus type 1 Thymidine kinase, Short synthetic: based on the highly efficient polyA signal of the rabbit beta-globin gene, SV40late: Viral Simian virus 40 late polyA terminator element, rBG or rbBG: rabbit beta-globin, hGH: human Growth Hormone.

Figure 13. PU.1, IRF8 and BATF3 allow high cDC1 reprogramming efficiency in human cancer cells independently of the order of the transcription factors in the tricistronic cassette. (A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding tricistronic cassettes containing the coding sequences of PU.1, IRF8 and BATF3 in different orders 3 days after transduction of glioblastoma T98G, melanoma A2058 and sarcoma SK-LMS-1 human cancer cell lines, and (B) Colorectal CRC50, head and neck ASG04 and Lung L6093 primary cancer samples using two different MOIs (100 and 1000 IFU/cell). Reprogramming efficiency was measured as percentage of transduced cells expressing CD45 and HLA-DR (n=2-6). Mean ± SD are represented.

Figure 14. Ad5-PIB vector with SFFV promoter, PIB polycistronic cassete, WPREmut6 and rbBG polyadenylation signal allows higher cDC1 reprogramming efficiency at low multiplicity of infection and superior efficacy. (A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding PU.1, IRF8 and BATF3 (PIB) followed by WPRE-BGHpA or WPREmut6-rbBGpA 3 days after transduction of glioblastoma T98G, melanoma A2058, sarcoma SK-LMS-1 and head and neck Ca9-22 human cancer cell lines, and (B) Colorectal CRC50 and head and neck ASG04 primary cancer samples using two different MOIs (100 and 1000 IFU/cell). Reprogramming efficiency was measured as percentage of transduced cells expressing CD45 and H LA-DR (n=2-11). Mean ± SD are represented. (C) Patient-derived cancer samples of melanoma (n=6 patients) were transduced with Ad5-PIB vectors with WPRE-BGHpA (Ad5-PIB) or WPREmut6-rbBGpA (AT-108) and reprogramming efficiency was evaluated by flow cytometry quantification of CD45 and HLA-DR, HLA-ABC, cDC1 markers CD141 and CLEC9A, and costimulatory molecules CD40, CD80 and CD86. (D) Flow cytometry quantification of CD45, HLA-DR and CD40 expression in patient-derived organoids of colorectal (n=2 patients) and head and neck (n=2 patients) cancer 3 days after transduction with Ad5- PIB or AT-108. (E) Human SK-LMS-1 cell line-derived xenografts were injected with Ad5- PIB or AT-108 at days 0, 2, 4 and 6, and in vivo reprogramming was evaluated by flow cytometry quantification of CD45 and HLA-DR expression in CD44+ SK-LMS-1 cells. (F) Primary melanoma cells transduced with Ad5-PIB or AT-108 at day 8 post transduction were pulsed with long MART-1 peptide and stimulated overnight with TLR agonists (TLR3 (Poly l:C), TLR4 (LPS) and TLR7/8 (R848)). Antigen cross-presentation was evaluated 8 days after co-cultured with HLA-A2+ MART-1+ CD8+ T cells in the presence of IL-2 and IL-7 (n=3 HLA-A2+ patients). (G) Primary melanoma cells transduced with Ad5-PIB or AT-108 at day 8 post transduction were stimulated overnight with TLR agonists (TLR3 (Poly l:C), TLR4 (LPS) and TLR7/8 (R848)), and cytokines (IL-12p70, TNFa and IFNB) in culture media were quantified using cytometric bead array (CBA). (H) Ad5-PIB or AT-108 were delivered via intra-tumoral injection in B16 tumors at days 0, 2, 4 and 6, and survival was profiled overtime. Mice received immune-checkpoint blockade with aPD-1 and a-CTLA-4 antibodies at days 0, 3 and 6.

Figure 15. Ad5/F35 allows higher transduction efficiency across human cancer cell lines and primary cancer cells at low multiplicity of infection. (A) Flow cytometry quantification of transduction efficiency mediated by eGFP-encoding adenoviral vectors serotype 5 (Ad5), serotype 5 with fiber and nob of Ad35 (Ad5/F35), and serotype 5 with RGD motif added in the nob (Ad5-RGD) 3 days after transduction of 4 human cancer cell lines (glioblastoma T98G, melanoma A2058, sarcoma SK-LMS-1 and head and neck Ca9-22) and 13 primary cancer samples (3 melanoma, 3 lung, 3 head and neck, 3 colorectal and 1 breast) using two different MOIs (100 and 1000 IFU/cell). Transduction efficiency was measured as percentage of eGFP+ transduced cells within live cells (n=2 per cell line or primary sample).

Figure 16. Ad5-RGD, Ad5/F35 and Ad5/3 allow higher reprogramming yield in human cancer cell lines and primary samples when compared to Ad5. (A) Flow cytometry quantification of cDC1 reprogramming yield mediated by adenoviral vectors serotype 5 (Ad5), serotype 5 with RGD motif added in the nob (Ad5-RGD), serotype 5 with fiber and nob of Ad3 (Ad5/3) and serotype 5 with fiber and nob of Ad35 (Ad5/F35) encoding PU.1, IRF8 and BATF3 (PIB) followed by WPREmut6-rbBGpA 3 days after transduction of glioblastoma T98G, melanoma A2058, sarcoma SK-LMS-1 and head and neck Ca9-22 human cancer cell lines, and (B) melanoma M2778, head and neck ASG04 and colorectal CRC50 primary cancer samples using three different MOIs (10, 100 and 1000 IFU/cell). Reprogramming yield was measured as percentage of live cells expressing CD45 and H LA-DR (n=2-6). # marks conditions where cell toxicity was observed. Mean ± SD are represented. Group comparisons were performed using two- way ANOVA and corrected by Tukey's multiple comparison test. *p < 0.05; **p < 0.01 ; ***p < 0.001; ****p < 0.0001 (C) Maps of the adenoviral vectors serotype 5 (Ad5), serotype 5 with RGD motif added in the nob (Ad5-RGD), serotype 5 with fiber and nob of Ad3 (Ad5/3), and serotype 5 with fiber and nob of Ad35 (Ad5/F35), encoding PU.1 , IRF8 and BATF3 (PIB) followed by WPREmut6-rbBGpA, (SEQ ID NOs: 1-4).

Figure 17. Lower TMB and MSS status associate with higher cDC1 reprogramming efficiency. Plots showing correlation between reprogramming efficiency measured as %CD45+ and/or HLA-DR+ live cells and tumor mutational burden (TMB, # non- synonymous mutations (number of non-synonymous mutations in the genome of the sample tested); left panel) or Microsatellite stability status (MSS: stable; MSI: Instable; right panel).

Figure 18. In vivo cDC1 reprogramming elicits tumor-origin agnostic immunity.

(A) mice were injected with cancer cells subcutaneously (CT26 colon cancer) or orthotopically into the left mammary fat pad (4T1 breast cancer), intracranially (SB28 glioblastoma) or intravenously (LLC lung cancer) after transduction with PIB-eGFP or control eGFP and mixing 1 :1 with parental cells to induce tumor cell reprogramming in vivo along with tumor establishment. Survival is shown for CT26, 4T1 and SB28 models (n = 5). For LLC, tumor area per lung area was quantified. Anti-PD-1 or isotype control antibodies were administered by intraperitoneal injection at days 7, 10, and 13 after tumor establishment. (B) Mice were injected with cancer cells subcutaneously with MC38 colon cancer and MB49 bladder cancer after transduction with Ad5-PIB or control Ad5-Stuffer to induce tumor cell reprogramming in vivo along with tumor establishment. Survival is shown (n=8).

Figure 19. In vivo cDC1 elicits systemic tumor-specific antibody production. (A) Flow cytometry quantification of the percentages of TIL-B cell populations including B cells, lgM+ B cells, lgG1+ B cells, activated B cells, mature B cells, plasma cells, lgG1 + plasma cells and follicular dendritic cells at day 21 after tumor establishment by subcutaneous injection of YUMM1.7 cells transduced in vitro with PIB-eGFP- and eGFP- encoding lentiviral vectors. (B) Flow cytometry quantification of Mean fluorescence intensity (MFI) of serum IgM, lgG1 and IgA antibodies bound to YUMM1.7 cells in vitro. Serum was isolated at indicated time-points (5, 9, 14, 21 and 28) from mice treated with sub-cutaneous injection of YUMM1.7 cells transduced in vitro with PIB-eGFP- or eGFP- encoding lentiviral vectors. (n=5-8).

Figure 20. Lowest dose of AT-108 at 108 viral particles per dose is sufficient to induce in vivo efficacy. (A) Subcutaneous mouse melanoma B16 tumors were treated with PBS (n=7) or AT-108 (n=8-9) via intratumoral injection at day 0, 2, 4, 9 and 14 at doses of 10^s, 10⁹ and 10¹° viral particles (VPs) of AT-108 per dose. Mice were also treated with anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. (B) Individual tumor growth kinetics over time after the first treatment. (C) Individual tumor growth volumes at day 14. (D) Kaplan-Meir survival analysis over time.

Figure 21. AT-108 as monotherapy treatment induce in vivo efficacy. (A) Subcutaneous mouse melanoma B16 tumors were treated with AT-108 (n=7) via intratumoral injection at day 0, 2, 4, 9 and 14 with 10¹⁰ viral particles per AT-108 dose or PBS (n=7-8). PBS groups were treated with or without anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. (B) Individual tumor growth volumes at day 10. (C) Individual tumor growth kinetics over time after the first treatment. (D) Kaplan-Meir survival analysis over time comparing PBS, PBS+ICB and AT-108 at a dose of 1O¹⁰ viral particles. (E) Kaplan-Meir survival over time comparing PBS and AT-108 groups receiving 10⁸, 10⁹or 10¹° viral particles per dose.

Figure 22. A single intratumoral injection of Ad5-PIB is sufficient to induce in vivo efficacy, further improved by multiple dosing. (A) Subcutaneous mouse melanoma B16 tumors were treated with AT-108 (n=8-17), Ad5-Stuffer (n=8-15) or PBS (n=5) via intratumoral injection 1 , 2, 3 or 4 times at 10¹⁰ viral particles per dose. Mice were also treated with anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. (B) Kaplan-Meir survival analysis over time.

Figure 23. Weekly booster cycles of AT-108 significantly improve survival. (A) Subcutaneous mouse melanoma B16 tumors were treated with Ad5-PIB (n=8-9) or Ad5-Stuffer (n=6) via intratumoral injection at day 0, 2, 4, 7 at 10¹⁰ viral particles per dose. Booster cycles was performed at week 2, 3, 4 and 5. Mice were also treated with anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. (B) Kaplan-Meier survival analysis over time.

Figure 24. In situ cDC1 reprogramming mediated by adenoviral vectors encoding PU.1, IRF8 and BATF3 induces local control of tumor growth and abscopal effect. Mice were subcutaneously inoculated with 200,000 murine B16 melanoma cells on both flanks. After both tumors emerged, one tumor was treated with adenoviral vectors encoding PU.1, IRF8 and BATF3 (Ad5-PIB, n=11) or a non-coding stuffer sequence (Ad5-Stuffer, n=9) at day 0 (at a tumor volume of 30-100 mm3), 2, 4, 6. Mice were also treated with anti-PD1 and anti-CTLA-4 antibodies (ICB) by intraperitoneal administration at day 0, 3 and 6. (A) Tumor growth kinetics of non-treated and treated tumors with Ad5- PIB or Ad5-Stuffer. Arrows indicate treatment timings. (B) Quantification of volume of non-treated and treated tumors in Ad5-PIB-treated mice at day 17. Complete Responders (CR), Partial Responders (PR). (C) Kaplan-Meier survival analysis over time. Mean ± SEM is represented, and statistical differences are shown when relevant (p**<0.01).

Figure 25. In situ cDC1 reprogramming with Ad5-PIB induces infiltration of immune cells in the treated and non-treated tumors. Flow cytometry quantification tumor-infiltrating (A) immune cells (CD45+), (B) CD8+ T cells, effector (T-bet+PD-1-), exhausted (T-bet+PD-1+) and terminally exhausted (T-bet-PD-1+), (C) CD4+ T cells, CD4+ Tregs (CD25+CD44+) and Th1/effector CD4+ T cells (CD25-CD44+T-bet+), (D) NK cells (CD3-NK1.1+) and (E) B cells (CD19+). (F) Correlation of frequency of CD8+, CD4+ and NK1.1 cells in the treated tumor compared to abscopal non-treated tumor.Statistical differences are shown when relevant (*p<0.05, **p<0.01, ***p<0.001 , ****p<0.0001).

Figure 26. In situ cDC1 reprogramming induces control of YUMM1.7 tumor growth. Murine YUMM1.7 melanoma cells were implanted via subcutaneous injection in C57BL/6J mice. Treatment was initiated when tumor volumes reached 30-100mm³ (day 0). Ad5-Stuffer and Ad5-PIB (1x10¹⁰ viral particles in PBS) or PBS only were administered via intratumoral injection at days 0, 2, 4 and 7 (four injections total). Intraperitoneal injections of aPD-1 (200 pg/mice) or IgG control in 100 ul of PBS were performed at days 0, 3 and 7 (three injections total). Tumor growth and survival were monitored. (A) YUMM1.7 melanoma growth curves and (B) survival of mice treated with PBS (black) and Ad5-PIB (grey) in monotherapy and in combination with aPD-1. (C) flow cytometry quantification of YUMM1.7-reactive CD4+ T cells after co-culture of PBMCs isolated from treated mice 10 days after first dosing. PBMCs were co-cultured overnight with IFN-Y-stimulated YUMM1.7 cells, and tumor reactivity was quantified by flow cytometry analysis of IFN-y+ CD4+ T cells.

Figure 27. AT-108 induces in vivo efficacy in immunosuppressed PANC02 model in combination with aPD-1 blockade. Murine PANC02 pancreatic cancer cells were implanted via subcutaneous injection in C57BL/6J mice. Treatment was initiated when tumor volumes reached 30-100mm³ (day 0). Ad5-Stuffer and Ad5-PIB (1x10¹° viral particles in PBS) or PBS only were administered via intratumoral injection at days 0, 2, 4 and 6 (four injections total). Intraperitoneal injections of aPD-1 (200 pg/mice) or IgG control in 100 ul of PBS were performed at days 0, 3 and 6 (three injections total). Tumor growth was evaluated at day 12 and compared between groups.

Figure 28. Inclusion of 2 maintenance cycles following intense 3-dose cycle enhances in vivo efficacy and extend median overall survival. Subcutaneous mouse melanoma B16 tumors were treated with AT-108 or PBS via intratumoral injection at day 0, 2, 4 at 10¹⁰ viral particles per dose. Maintenance cycles (booster cycles) were performed at days 9 and 14. Mice were also treated with anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. Kaplan-Meier survival analysis was performed for both groups receiving the 3-dose lead cycle and 3-dose lead cycle with 2 maintenance cycles (booster cycles). Median overall survival (OS) is highlighted in the Kaplan-Meier plots and in the bottom of the figure.

Figure 29. Less intense AT-108 dosing allows in vivo efficacy in combo with ICB.

Subcutaneous mouse melanoma B16 tumors were treated with AT-108 or PBS via intra-tumoral injection at day 0, 2, 4, 9 and 14, or at days 0, 5, 10 and 15 at 10¹⁰ viral particles per dose. Mice were also treated with anti-PD-1 and anti-CTLA-4 (ICB) via intraperitoneal injection at day 0, 3 and 7. Kaplan-Meier survival analysis was performed for both groups receiving the 3-dose lead cycle with 2 maintenance cycles (booster cycles) and the 4-dose cycle with injections every 5 days. Median overall survival (OS) is highlighted in the Kaplan-Meier plots and in the bottom of the figure.

Figure 30. Intraperitoneal administration of AT-108 induces anti-tumor efficacy in the ID8 ascites model as monotherapy. (A) Intra-peritoneal mouse ovarian ID8 ascites were treated with AT-108 or PBS via intra-tumoral injection at day 0, 2, 4, 9 and 14 at 3x10¹⁰viral particles per dose. Mice were also treated with anti-PD-1 via intraperitoneal injection at day 0, 3 and 7. Therapeutic efficacy was determined by longitudinal luciferase radiance monitoring. (B) Quantification of luciferase radiance from day zero (baseline) to day 36 post first dose. Statistical analyses was performed using data collected on Day 36 post-randomization.

Figure 31. AT-108 induces dose-dependent functional cDC1 reprogramming in patient-derived samples. (A) Flow cytometry quantification of reprogramming efficiency in patient-derived melanoma (n=6 patients), head and neck (n=4 patients) and colorectal cancer (n=9 patients) 9 days after transduction with AT-108 at different multiplicities of infection (MOI, infective units (IFU) per cell). Reprogramming efficiency was measured by the frequency of cancer cells expressing CD45 and/or HLA-DR. Frequency of transduced melanoma cells expressing cDC1 markers (CLEC9A, CD141 and CD11C), MHC-I (HLA-ABC) and co-stimulatory molecules (CD40, CD80 and CD86) was also quantified. (B) Flow cytometry quantification of reprogramming efficiency in patient-derived organoids of head and neck (n=3 patients) and colorectal cancer measured as frequency of cells expressing CD45 and/or HLA-DR. (C) Quantification of cytokine secretion capacity by primary melanoma cells (n=6 donors) 9 days after transduction with AT-108 at different MOIs, with or without overnight TLR stimulation. (D) Quantification of antigen cross-presentation capacity of transduced primary melanoma cells (n=3 melanoma patients) measured as frequency of MART-1 + CD8+ T cells 8 days after co-culture. (E) Flow cytometry quantification of allogeneic T cell activation 1 day (IFNy+) or 3 days (CD69+ and PD-1+) after co-culture with transduced primary cancer cells of head and neck (n=3 patients) and colorectal cancer (n=4 patients). (F) 3 primary melanoma samples were transduced with AT-108 at increasing MOI and seeded in E-plates compatible with the xCELLigence system. Autologous tumor-infiltrating lymphocytes (TILs) were added and co-cultured for 72 hours with transduced cancer cells. Real-time impedance measurements were recorded throughout the assay, and % tumor cell cytolysis was quantified, reflecting target cell killing.

Detailed description

Definitions

“biologically active variant” refers herein to a biologically active variant of a genetic element such as of a regulatory element, of a transcription factor (TF), or of a reprogramming modulator, which retains at least some of the functional activity of the parent genetic element, TF or reprogramming modulator. The term encompasses variants at the polypeptide level, including protein isoforms, that exhibit a minimum of 90% sequence similarity to the parent sequence. These variants may differ in their efficiency of inducing or inhibiting gene expression compared to the parent TF or modulator. For example, a biologically active variant of Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), Interferon Regulatory Factor 8 (IRF8), and PU.1 can act as said respective TF and induce or inhibit expression of the same genes in a cell as BATF3, IRF8, and PU.1, respectively, although the efficiency of the induction may be different, e.g. the efficiency of inducing or inhibiting genes is decreased or increased compared to the parent TF.

“Identity” and “homology”, with respect to a polynucleotide or polypeptide, are defined herein as the percentage of nucleic acids or amino acids in the candidate sequence that are identical or homologous, respectively, to the residues of corresponding native nucleic acids or amino acids, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity / similarity / homology, and considering any conservative substitutions according to the NCIUB rules (htp://www.chem. qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Generally, a given homology between two sequences implies that the identity between these sequences is at least equal to the homology; for example, if two sequences are 70% homologous to one another, they cannot be less than 70% identical to one another - but could be sharing 80% identity.

" urine" refers herein to any and all members of the family Muridae, including rats and mice.

“Reprogramming” refers herein to the process of converting of differentiating cells from one cell type into another. In particular, reprogramming herein refers to converting or transdifferentiating any type of cell into a dendritic cell, such as a conventional type 1 dendritic cell (cDC1), or an antigen-presenting cell.

“affiliation” as used herein refers to the classification or assignment of individual cells into specific categories or groups based on their gene expression profiles. The affiliation can be performed for instance by the methods described herein, for example involving using a support vector machine (SVM) classifier to predict or determine the identity or type of cells (such as dendritic cells) based on patterns found in their gene expression profiles as compared to known reference datasets.

“Treating,” or “Treatment,” refers herein to any administration or application of a therapeutic for the disclosed diseases, disorders and conditions in subject, and includes inhibiting the progression of the disease, slowing the disease or its progression, arresting its development, partially or fully relieving the disease, or partially or fully relieving one or more symptoms of a disease.

As used herein, the term "adenovirus" is used to refer to any and all viruses that may be categorized as an adenovirus, including any adenovirus that infects a human or a non-human animal, including all groups, subgroups, and serotypes, except when required otherwise. Thus, as used herein, "adenovirus" refers to the virus itself or derivatives thereof and cover all serotypes and subtypes, naturally occurring (wildtype), modifications to be used as an adenoviral vector, e.g., a gene delivery vehicle, forms modified in ways known in the art, such as for example capsid mutations, and recombinant forms, replication-competent, conditionally replication-competent, or replication-deficient forms, except where indicated otherwise.

As used herein, the term “adeno-associated virus” may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term is used to refer to any and all viruses that may be categorized as an adeno-associated virus, including any adeno-associated virus that infects a human or a non-human animal, and covers all subtypes, serotypes and pseudotypes, and both naturally occurring, modified and recombinant forms, such as modifications to be used as an adeno-associated viral vector, e.g., a gene delivery vehicle except where required otherwise.

As used herein, unless otherwise specified e.g. in figure legends, the abbreviation "Ad" in the context of a viral vector refers to an adenoviral vector and is typically followed by a number indicating the serotype of the adenovirus. For example, "Ad5" refers to adenovirus serotype-5 vectors.

The term “Ad”, when not followed by a specific number, covers any Ad suitable for the purpose may be used herein, such as but not limited to Ad from any serotype from any of the A, B, C, D, E, F, G Ad subgroups, for example Ad2, Ad5, or Ad35, avian Ad, bovine Ad, canine Ad, caprine Ad, equine Ad, primate Ad, nonprimate Ad, and ovine Ad. "Primate Ad refers to Ad that infect primates, "non-primate Ad" refers to Ad that infect non-primate mammals, "bovine Ad” refers to Ad that infect bovine mammals.

The genomic sequences of various serotypes of Ad, as well as the sequences of the native terminal repeats (TRs) and capsid subunits are known in the art.

As used herein, the abbreviation “AAV” in the context of a viral vector refers to an adeno-associated virus and is typically followed by a number indicating the serotype of the adeno-associated virus. For example, "AAV2" refers to adeno-associated virus serotype 2.

The term covers any suitable AAV, such as but not limited to AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3A (AAV3A), AAV serotype 3B (AAV3B), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, nonprimate AAV, and ovine AAV. "Primate AAV refers to AAV that infect primates, "nonprimate AAV" refers to AAV that infect non-primate mammals, "bovine AAV refers to AAV that infect bovine mammals.

The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art.

“Hybrid” or “chimeric” Ad or AAV vectors as used herein refers to vectors based on Ads or AAVs engineered in a way that the Ad or AAV vectors contains proteins derived from two or more different Ad or AAV serotypes. Ad5/3 and Ad5/F35 as described herein are examples of such hybrid or chimeric Ad vectors.

“AAV2-qYF” or “AAV2-QuadYF” as used herein refers to a quadruple tyrosine to phenylalanine mutant of AAV2.

“AAV-DJ” as used herein refers to a hybrid capsid derived from DNA family shuffling of 8 wild type serotypes of AAV, including AAV 2, 4, 5, 8, 9, avian, bovine and caprine AAV. AAV-DJ is a synthetic serotype, type 2/type 8/type 9 chimera, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids.

“rBGpA” or “rbBGpA” as used herein refer to the Rabbit beta-globin polyadenylation signal.

“SV40late” as used herein refers to the Viral Simian virus 40 late polyA terminator element.

“WPREmut6” as used herein refers to the mutated Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element sequence carrying a mutation disturbing the expression of a truncated Woodchuck hepatitis virus X protein implicated in liver tumors, as first described in Kingsman et al. 2005.

As used herein, the terms “booster cycles” and “maintenance cycles” will be used interchangeably. Constructs

One aspect of the invention relates to one or more constructs, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1 , IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40).

In some preferred embodiments, one of the at least two transcription factors is PU.1 .

In some embodiments, the one or more constructs, upon expression, encode PU.1 , or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 10, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 10.

PU.1 may thus preferably be the human PU.1, such as human PU.1 protein isoform 1 with accession number NP_003111 .2.

In other embodiments, the one or more constructs, upon expression, encode IRF8, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 12, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 12.

IRF8 may thus preferably be the human IRF8, such as human IRF8 protein isoform 1 with accession number NP_001350836.1 or human IRF8 protein isoform 1 isoform 2 with accession number NP_003111.2.

In some embodiments, the one or more constructs, upon expression, encode BATF3, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 14, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 14.

BATF3 may thus preferably be the human BATF3, such as human BATF3 with accession number NP_061134.1.

The one or more constructs of the present invention may be polycistronic or monocistronic, for example each construct may encode 1 , 2, or 3 transcription factors, with the polynucleotide sequence encoding the last transcription factor (3’ end of the construct) being further followed by a stop codon to terminate translation, preferably the stop codon TGA.

Thus, in embodiments of the present invention, the one or more constructs comprise: a) one construct which upon expression encodes the transcription factors PU.1 , IRF8 and BATF3; b) one construct which upon expression encodes the transcription factors IRF8 and BATF3; c) one construct which upon expression encodes the transcription factors PU.1 and BATF3; d) one construct which upon expression encodes the transcription factors PU.1 and IRF8; e) a first construct which upon expression encodes the transcription factors IRF8 and BATF3, and a second construct which upon expression encodes the transcription factor PU.1 ; f) a first construct which upon expression encodes the transcription factor BATF3, and a second construct which upon expression encodes the transcription factors PU.1 and IRF8; g) a first construct which upon expression encodes the transcription factor IRF8, and a second construct which upon expression encodes the transcription factors PU.1 and BATF3; and/or h) a first construct which upon expression encodes the transcription factor PU.1 ; a second construct which upon expression encodes the transcription factor IRF8; and a third construct which upon expression encodes the transcription factor BATF3. In some embodiments of the one or more constructs of the present invention, PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 9 PU.1 may thus preferably be the human PU.1 , such as human PU.1 transcript variant 2 encoded by the sequence set forth in accession number NM_003120.3.

The skilled person will know that it may be beneficial to introduce mutations in constructs in order to facilitate for example cloning and/or vector manufacturing while keeping the functionality of the expressed factors. For example, silent or neutral mutations may be introduced in the constructs in order to remove restriction sites at unwanted locations in the constructs, which may for instance impair the manufacturing of the constructs or vectors comprising thereof.

The constructs of the present invention may thus comprise silent or neutral mutations removing restriction sites, such as SFi 1 restriction sites, for example in the SFi 1 restriction site located at the 3’ end of the human native PU.1 sequence isoform 2 encoded by the sequence set forth in accession number NM_003120.3.

A skilled person would also recognize that it may be advantageous to co-express additional transgenes alongside PU.1 , IRF8, and BATF3. For instance, Ziblat et al. have shown that conventional type 1 dendritic cells (cDC1s) can promote the reinvigoration of CD8⁺ T cells through 4-1 BB/4-1 BBL signaling, particularly when PD- 1/PD-L1 checkpoint pathways are inhibited. This interaction enhances the therapeutic efficacy of PD-1/PD-L1 blockade. Therefore, a logical and beneficial transgene to include in the same vector as PU.1, IRF8, and BATF3 would be TNFSF9, which encodes the 4-1 BB ligand (4-1 BBL).

In other embodiments of the one or more constructs of the present invention, IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 11. IRF8 may thus preferably be the human IRF8, such as human IRF8 transcript variant 1 encoded by the sequence set forth in accession number NM_001363907.1 and human IRF8 transcript variant 2 encoded by the sequence set forth in accession number NM_003120.3. In further embodiments of the one or more constructs of the present invention, BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 13.

BATF3 may thus preferably be the human BATF3, such as human BATF3 encoded by the sequence set forth in accession number NM_018664.3.

In some embodiments of the one or more constructs described herein, the at least two transcription factors are in the following sequential order from 5’ to 3’:

PU.1 , IRF8, BATF3;

PU.1 , BATF3, IRF8;

IRF8, PU.1 , BATF3;

IRF8, BATF3, PU.1;

BATF3, PU.1 , IRF8; or

BATF3, IRF8, PU.1.

In some embodiments of the one or more constructs of the present invention, the sequential order of transcription factors from 5’ to 3’ is PU.1 , IRF8, BATF3.

In preferred embodiments, such as for polycistronic constructs, the polynucleotide sequence encoding the last transcription factor (3’ end of the construct) is further followed by a stop codon to terminate translation, preferably the stop codon TGA.

In preferred embodiments of the one or more constructs of the present invention, the SFFV promoter comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 15, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 15, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 15.

In other preferred embodiments of the one or more constructs of the present invention, the mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 6, or a biologically active variant thereof a polynucleotide sequence having at least 90% identity to SEQ ID NO: 6, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 6

In yet other embodiments of the one or more constructs of the present invention, the rabbit beta-globin polyadenylation signal sequence (rbBGpA) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 7, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 7, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 7.

In embodiments of the one or more constructs of the present invention, the late polyadenylation signal sequence of simian virus 40 (SV40late) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 8, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 8, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 8.

The one or more constructs of the present invention may in some embodiments comprise one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element sequence (WPREmut6), or a variant thereof, having at least 90% identity to SEQ ID NO: 6, the rabbit beta-globin polyadenylation signal sequence (rbBGpA), or a variant thereof, having at least 90% identity to SEQ ID NO: 7, and the late polyadenylation signal sequence of simian virus 40 (SV40late), or a variant thereof, having at least 90% identity to SEQ ID NO: 8.

Thus, in some embodiments of the present invention, the one or more sequences are: a) WPREmut6; b) rbBGpA; c) SV40late; d) WPREmut6 and rbBGpA; e) WPREmut6 and SV40late; or f) rbBGpA and SV40late. Constructs with expression cassettes containing WPREmut6 and rbBGpA have for instance been surprisingly shown in the present invention to increase cell reprogramming efficiency compared to constructs with expression cassette containing WPRE-BGHpA, such to increase cancer cell reprogramming to cDC1 cells.

Interestingly, the inventors have found in the present invention that constructs comprising rbBGpA were able to increase the percentage of reprogrammed cDC1 cells across several cell types, and already at low MOI, such as 100 to 500 I FU/cell. Further, for some applications, it may be beneficial that the polyadenylation signal sequence originates from a mammalian organism.

Thus, in preferred embodiments, the one or more constructs comprise or consist of: SFFV, PU.1 , IRF8, BATF3, WPREmut6, and rbBGpA.

The inventors have identified in the present invention construct elements which alone, but even more surprisingly also combined, enhance the reprogramming efficiency and/or fidelity of cells to cDC1 cells.

Thus, in other preferred embodiments, the one or more constructs of the present invention comprise or consist of, in sequential order from 5’ to 3’: SFFV, PU.1, IRF8, BATF3, WPREmut6, and rbBGpA.

In other preferred embodiments, the one or more constructs of the present invention further comprise self cleaving peptides or 2A peptides.

In even more preferred embodiments, the one or more constructs of the present invention thus comprise or consist of, in sequential order from 5’ to 3’: SFFV, PU.1 , 2A peptide, IRF8, 2A peptide, BATF3, WPREmut6, and rbBGpA, such as SFFV, PU.1 , P2A, IRF8, T2A, BATF3, WPREmut6, and rbBGpA, preferably wherein the P2A and T2A peptides or variants thereof, are encoded by polynucleotide sequences comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 28 and SEQ ID NO: 29, respectively, or variants thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

The inventors have optimized a cassette expressing reprogramming transcription factors, comprising the SFFV, PU.1 , P2A peptide, IRF8, T2A peptide, BATF3, WPREmut6, and rbBGpA elements which separately but even more unexpectedly increase reprogramming efficiency as supported by the Examples herein.

Thus, in even more preferred embodiments, the one or more constructs comprise or consist of the polynucleotide sequence set forth in SEQ ID NO: 5, or a variant thereof having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5

Vectors

The constructs of the present invention find applications in the field of, but not limited to, gene therapy and cell therapy. For these applications, and other relevant applications, it may be beneficial that said constructs are integrated in vectors, such as vectors comprising further elements useful for gene delivery, expression, stability.

Techniques for producing vectors, such as adenoviral vectors comprising defined constructs are known to the skilled person, and typically involve cloning the constructs comprising the gene(s) of interest into a plasmid or cosmid vector and recombination with a viral vector backbone such as an adenoviral backbone, transfection in host cells such as HEK293 cells for packaging, and amplification of adenoviral particles, followed by purification of the recombinant adenoviral particles and quality control. The skilled person will appreciate that further elements known in the art to facilitate vector manufacturing may be added to said vectors, such as multiple cloning sites, for example a multiple cloning site (MCS) inserted in place of the E3 adenoviral region, such as a 69-bp MCS sequence (SEQ. ID NO 34) inserted in place of the E3 region between the Xbal sites located in the Ad5 E3 region, thereby deleting 1 .9 kb from the genome.

The skilled person will appreciate that the identification of optimized combinations of construct and vector elements, such as for cell reprogramming applications, is not straightforward due to the complex interplay of between the components of the construct (promoter, transcription factors, regulatory elements) with the vector, and host cell environment. This is a fortiori also the case for in vivo applications of said vectors.

In another aspect, the present invention provides one or more vectors comprising the one or more constructs of the present disclosure. As mentioned in the section “Constructs” above, the one or more constructs of the present invention may be polycistronic or monocistronic, for example each construct may encode 1, 2, or 3 transcription factors, such as PU.1, IRF8 and/or BATF3. Therefore in some embodiments, the one or more vectors comprising the one or more constructs of the present disclosure may be for example one vector comprising one construct encoding the transcription factors PU.1 , IRF8 and BATF3, or more than one vectors together encoding PU.1 , IRF8 and BATF3, such as 2 vectors, such as 3 vectors.

In some embodiments, the one or more vectors is a viral vector.

In other embodiments, the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retrovirus vectors, herpes virus vectors, pox virus vectors, adeno-associated virus vectors, paramyxoviridae vectors, rabdoviral vectors, alphaviral vectors, flaviral vectors, and adeno-associated viral vectors.

In preferred embodiments, the viral vector is an adenoviral (Ad) vector.

In other embodiments, the adenoviral vector is selected from the group consisting of: wild-type Ad vectors, chimeric Ad vectors, and mutant Ad vectors.

In further embodiments, the wild-type Ad vector is Ad5.

In other preferred embodiments, the Ad vector is selected from the group consisting of: Ad5-RGD, Ad5/F35 and Ad5/3, preferably wherein the Ad vector is Ad5/F35, even more preferably wherein the Ad vector is Ad5 or Ad5-RGD.

In other embodiments, the viral vector is a lentiviral vector.

In further embodiments, the adeno-associated virus vector is selected from the group consisting of : wild-type AAV vectors, hybrid AAV vectors and mutant AAV vectors.

In some embodiments, the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV2-QuadYF. Similarly to restriction sites present within the construct sequences, for some applications, it may be preferable that native restriction sites present on the vector sequence and targeted by restriction enzymes are mutated, for example to facilitate proper cloning of the vector elements, such as directional cloning. Thus in some embodiments of the vectors of the present invention, one or more Sfil sites have been mutated, preferably by silent mutations, even more preferably wherein said Sfil sites are in the pVII ORF and/or the adenovirus DNA-binding protein (DBP) ORF the vector.

In preferred embodiments of the one or more vectors of the present invention, the vector comprises an Ad5 wild-type fiber. The skilled person will appreciate that cellular internalization of vectors comprising an Ad5 wild-type fiber is mediated by the cell surface coxsackievirus and adenovirus receptor (CAR).

Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 1 , or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1.

In other preferred embodiments, the vector Ad5 wild-type fiber is replaced by the fiber of Ad35 (Ad5/F35). The skilled person will appreciate that cellular internalization of chimeric Ad5/F35 vectors is mediated by the CD46 receptor. Ad5/F35 and Ad5/35 are used herein as synonyms.

Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 2.

In other preferred embodiments, the vector Ad5 wild-type fiber is modified by incorporating an RGD (arginine-glycine-aspartic acid) motif into the fiber knob protein of said vector (Ad5-RGD). The skilled person will appreciate that cellular internalization of Ad5-RGD vectors is mediated by the CD51 receptor (integrin av), such as by the CD51 subunit of av 3 and avp5 integrins. Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 3.

In yet other preferred embodiments, the vector Ad5 wild-type fiber knob is replaced by an Ad3 fiber knob (Ad5/3). The skilled person will appreciate that cellular internalization of Ad5/3 vectors is mediated by CD46 and desmoglein-2 (DSG-2).

Thus in some embodiments of the one or more vectors of the present invention, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 4.

The skilled person will appreciate that intermediate vectors, such as plasmid or cosmid vectors are typically required for the production of adenoviral vector final products, Thus in some embodiments, the vectors of the present invention further comprise cosmid and /or plasmid elements, such as a lambda phage cos sequence, and/or lambda phage scrambled sequences.

Thus in some embodiments, the one or more vectors is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 30, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 30.

In other embodiments, the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 31 , or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 31.

In further embodiments, the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 32, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 32.

In yet other embodiments, the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 33, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 33.

Method of manufacturing the vectors

In a further aspect, the present invention provides a method of manufacturing the one or more vectors described herein, said method comprising: a) Providing a host cell capable of being transfected with a nucleic acid sequence encoding the one or more vectors, such as the adenoviral vectors described herein, for example the vectors of the second or seventeenth aspect of the present disclosure; b) Transfecting the host cell with the nucleic acid sequence encoding said vectors, preferably wherein the nucleic acid sequence comprises the one or more constructs described herein, even more preferably wherein the nucleic acid sequence comprises the one or more constructs of the first, fifteenth or sixteenth aspect of the present disclosure, yet even more preferably wherein the nucleic acid sequence comprises the one or more constructs comprising or consisting of the polynucleotide sequence of SEQ ID NO: 5, or a variant thereof having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5; c) Culturing the transfected host cell under conditions suitable for expression and assembly of the vectors, such as adenoviral particles; d) Harvesting the vectors from the cultured host cells; and e) Purifying the harvested vectors. Methods of manufacturing vectors, such as adenoviral vectors, are well-established in the field of molecular biology and biotechnology. The skilled person is familiar with various techniques used in each step of vector production.

The skilled person will commonly utilize established cell lines suitable for viral vector production, ensuring compatibility with the intended vector expression system, such as the HEK293, “CEVEC's Amniocyte Production cell line” (CAP), 293SF, A549, HeLa cell lines, and known derivatives thereof.

Standard transfection methods, include calcium phosphate precipitation, lipid-mediated transfection, electroporation, or viral transduction

The skilled person will know that culturing conditions are optimized to promote vector expression and assembly, such as media composition, incubation temperature, CO2 levels, and culture duration to support the host cell and vector expression requirements.

Harvesting of the vectors may be performed using cell lysis, filtration, or ultracentrifugation to harvest adenoviral particles or other vectors from the cultured host cells. The skilled person will know that important parameters for harvesting are yield and integrity of the vectors.

Several vector purification techniques are known in the art, including chromatography (e.g., ion exchange, size exclusion), centrifugation, and filtration.

Cells comprising the one or more constructs or vectors

For some applications, it may be beneficial to for instance generate and/or isolate cells which comprise the one or more constructs or the one or more vectors of the present invention, such as cells which have internalized the constructs of the present invention and/or which have been transduced by the vectors of the present invention. Said cells may for example not be fully-reprogrammed cells. Said cells may also be cells which already overexpress the transcription factors present in the expression cassette internalized. These cells may thus also be referred to as construct or vector host cells.

Thus, another aspect of the present invention provides a cell comprising the one or more constructs or the one or more vectors of the present disclosure. In some embodiments, the cell comprises: a) one construct or vector which upon expression encodes the transcription factors PU.1, IRF8 and BATF3; b) one construct or vector which upon expression encodes the transcription factors IRF8 and BATF3; c) one construct or vector which upon expression encodes the transcription factors PU.1 and BATF3; d) one construct or vector which upon expression encodes the transcription factors PU.1 and IRF8; e) a first construct or vector which upon expression encodes the transcription factors IRF8 and BATF3, and a second construct or vector which upon expression encodes the transcription factor PU.1 ; f) a first construct or vector which upon expression encodes the transcription factor BATF3, and a second construct or vector which upon expression encodes the transcription factors PU.1 and IRF8; g) a first construct or vector which upon expression encodes the transcription factor IRF8, and a second construct or vector which upon expression encodes the transcription factors PU.1 and BATF3; and/or h) a first construct or vector which upon expression encodes the transcription factor PU.1 ; a second construct or vector which upon expression encodes the transcription factor IRF8; and a third construct or vector which upon expression encodes the transcription factor BATF3.

In preferred embodiments, the transcription factors are as defined in the present invention, for example in terms of the sequences and /or combinations described herein for the constructs and vectors of the present invention.

Thus, in preferred embodiments, the one or more constructs comprised in the cells are as defined herein. In other preferred embodiments, the one or more vectors comprised in the cells are as defined herein.

In some embodiments, the cell is a mammalian cell. In other embodiments, the cell is a human cell. In yet other embodiments, the cell is a murine cell. In other embodiments, the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell.

In some embodiments, the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a hematopoietic stem cell.

In preferred embodiments the differentiated cell is any somatic cell.

In other embodiments, the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte.

The skilled person will appreciate that cell-surface receptors can play an important role in the internalization of genetic material, such as constructs or vectors. The internalization mechanisms may take advantage of non-specific mechanisms and/or highly specific affinity for the construct or vector, such as receptor-mediated endocytosis, clathrin-dependent or -independent pathways, caveolin-mediated pathways, macropinocytosis, phagocystosis, or direct fusion with the membrane.

It may be preferable, for example in applications in which it is intended to transduce cells with viral vectors that specific receptors facilitating the entry of the viral particles into the host/target cells are present on said cells.

Thus, in some embodiments, the cell is expressing one or more of the surface markers selected from the group consisting of: Coxsackie and Adenovirus Receptor (CAR), CD51 , CD46 and DSG-2.

Method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell

Another aspect of the present invention provides a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, comprising the following step: a) transducing a cell with the one or more constructs of the first aspect of the present invention or the one or more vectors of the second aspect of the present invention. A further aspect of the invention provides a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, comprising the following steps: a) transducing a cell with the one or more constructs of the first aspect of the present invention or the one or more vectors of the second aspect of the present invention; b) expressing the transcription factors whereby a reprogrammed or induced cell is obtained.

In some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more cytokines, or contacting the cell with one or more cytokines.

In other embodiments, the one or more cytokines are pro-inflammatory cytokines.

In some embodiments, the one or more cytokines are hematopoietic cytokines.

In yet other embodiments, the one or more cytokines are selected from the group consisting of: IFNp, IFNy, TNFa, IFNa, IL-1 p, IL-6, CD40I, Flt3l, GM-CSF, IFN-A1 , IFN- co, IL-2, IL-4, IL-15, prostaglandin 2, SCF and oncostatin M (OM).

In some embodiments, the one or more cytokines are selected from the group consisting of: I FN|3, IFNy and TNFa.

In further embodiments of the method, the transducing step further comprises at least one vector comprising nucleic acids encoding immunostimulatory cytokines and/or siRNA targeting anti-inflammatory cytokines, including but not restricted to IL-10.

In other embodiments, the one or more methylation inhibitor is Azacitidine (Aza).

For example, the step of culturing or contacting the cells with valproic acid or azacitidine individually may be beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, melanoma cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells. In some preferred embodiments, the treatment with epigenetic modifiers comprises a combination of valproic acid and azacitidine. For example, this combination is beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, ovarian cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells.

For some applications, it may be preferable that the cell transduced in the methods of the present invention belongs to a specific cell type. This may be the case for instance in order to match the cell type with the organism from which the transcription factors expressed by the constructs or vectors of the present disclosure originate, and/or to improve the transfer and/or injection of the constructs or vectors, or the cells of the present invention with a receiving organism or subject.

In some embodiments, the cell is a mammalian cell.

In preferred embodiments, the cell is a human cell.

In other embodiments, the cell is a murine cell.

In embodiments of the method of the present invention, the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell.

In other embodiments, the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a mesenchymal stem cell and a hematopoietic stem cell.

In further embodiments, the differentiated cell is any somatic cell.

In preferred embodiments, the cell is expressing one or more of the surface markers selected from the group consisting of: Coxsackie and Adenovirus Receptor (CAR), CD51 , CD46, and DSG-2. In some embodiments, the transduced cell is cultured during at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

In embodiments of the method, the resulting reprogrammed or induced cell is a type 1 conventional dendritic cell.

In other embodiments, the resulting reprogrammed or induced cell is cluster differentiation 45 (CD45) positive.

In further embodiments the resulting reprogrammed or induced cell is human leukocyte antigen-DR isotype (HLA-DR) positive, preferably CD45 and HLA-DR positive.

Reprogrammed or induced cells obtained by the methods of the present invention In another aspect, the invention provides a reprogrammed or induced cell obtained by the method for reprogramming or inducing a cell into a dendritic cell or antigen- presenting cell as disclosed herein.

In preferred embodiments, the reprogrammed or induced cell is a dendritic or antigen- presenting cell, such as a type 1 conventional dendritic cell.

The generation of pure populations of cDC1 cells with high fidelity, as measured by cDC1 -specific marker expression is a challenge in the field. The present invention provides a solution to these challenges.

In further embodiments, the reprogrammed or induced cell cell is cluster differentiation 45 (CD45) positive.

In yet other embodiments, the reprogrammed or induced cell is cluster differentiation 226 (CD226) positive.

In other embodiments, the cell is human leukocyte antigen DR isotype (HLA-DR) positive. In some embodiments, the reprogrammed or induced cell is CD45, HLA-DR, CD141 , CLEC9A, XCR1 and/or CD226 positive, for example CD45- and HLA-DR-positive.

Pharmaceutical compositions

Another aspect of the invention provides a pharmaceutical composition comprising the one or more constructs, the one or more vectors, the cell, or the reprogrammed or induced cell according to the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In preferred embodiments, the pharmaceutical composition is for intratumoral administration, systemic administration such as via intravenous administration, intraperitoneal administration, or subcutaneous administration.

Medical uses and methods of treatment

In another aspect, the present invention provides the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein and/or the reprogrammed or induced cell as described herein, for use in medicine.

In a further aspect, the present invention provides the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell as described herein, for use in the treatment of cancer, such as solid tumor cancers and/or hematological cancers.

In embodiments of the uses described herein, the cancer is selected from the group consisting of: colorectal cancer, head and neck cancer, melanoma, breast cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, a germ cell tumor, a retinoblastoma, an age-related macular degeneration, glioblastoma, lymphoma, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, oesophageal cancer, uterus cancer, vaginal cancer, gastric cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, bladder and urothelial cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, skin cancer, and myeloma.

In embodiments of the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell for use as described herein, the cancer is selected from the group consisting of: colorectal cancer, head and neck cancer, melanoma, breast cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, a germ cell tumor, a retinoblastoma, an age-related macular degeneration, glioblastoma, lymphoma, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, oesophageal cancer, uterus cancer, vaginal cancer, gastric cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, bladder and urothelial cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, skin cancer, and myeloma.

In the present disclosure, inventors showed that lower tumor mutational burden (TMB) and microsatellite stability status associate with higher reprogramming efficiency.

Thus, in other embodiments of the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell for use as described herein, the cancer is a cancer having a low tumor mutational burden (TMB) such as metastatic microsatellite stable colorectal cancer, compared to microsatellite instable colorectal cancer.

Low tumor mutational burden (TMB) refers to a cancer or cancer cell characterized by a relatively low number of non-synonymous mutations within the tumor genome compared to another cancer or cancer cell. Low TMB may be defined as having no more than a certain number of non-synonymous mutations in the coding regions of the tumor genome. Non-synonymous mutations are mutations resulting in an alteration of an amino acid in the sequence of a protein and include missense mutations, insertions, deletions.

For example, in certain embodiments, a low TMB may be observed in cancers such as metastatic microsatellite stable (MSS) cancer, in contrast to cancers with high TMB such as microsatellite instable (MSI) cancer, which typically exhibit higher mutation frequencies.

In preferred embodiments, the cancer exhibiting microsatellite instability is selected from the group consisting of: colorectal cancer, brain cancer , melanoma, head and neck, lung cancer, and bladder cancer

In some embodiments, the low TMB threshold is measured through sequencing methods, such as sequencing of cancer tissue sample or cancer cells.

In preferred embodiments, the low tumor mutational burden (TMB) is characterized by at the most 2000 non-synonymous mutations, such as at the most 1500 non- synonymous mutations, such as a the most 1000 non-synonymous mutations.

In preferred embodiments, TMB is measured using whole-exome sequencing (WES) data, such as described herein in Example 18. In other preferred embodiments, MSI is measured by using mutations to infer the proportion of SBS6, SBS14, SBS15, SBS20, SBS21 , SBS26 and SBS44 MSI-associated mutation signatures (COSMIC: Catalogue of Somatic Mutations in Cancer; https://www.cosmickb.org) in each sample. In further preferred embodiments, samples are qualified as MSS if the total contribution of MSI- associated signatures is < 20, samples are qualified as MSI if the total contribution of MSI-associated signatures is > 25, and samples are given an unknown MSI status if the total contribution of MSI-associated signatures is comprised between 20 and 25.

In some preferred embodiments of the uses described herein, the cancer is selected from the group consisting of: melanoma, breast cancer, head and neck cancer, such as head and neck squamous cell carcinoma (HNSCC), such as head and neck squamous cell carcinoma (HNSCC) with combined positive score of PD-L1 less than 1 or HNSCC PD-L1 negative, sarcoma, colorectal cancer, such as metastatic microsatellite stable colorectal cancer.

In some embodiments, the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, head and neck cancer, colorectal cancer, sarcoma, liver cancer, and ovarian cancer. In some preferred embodiments of the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell for use as described herein, the cancer is selected from the group consisting of: melanoma, breast cancer, head and neck cancer, such as head and neck squamous cell carcinoma (HNSCC), such as head and neck squamous cell carcinoma (HNSCC) with combined positive score of PD-L1 less than 1 or HNSCC PD-L1 negative, sarcoma, colorectal cancer, such as metastatic microsatellite stable colorectal cancer. In some embodiments, the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, head and neck cancer, colorectal cancer, sarcoma, liver cancer, and ovarian cancer.

In yet other embodiments, the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, and head and neck cancer.

In preferred embodiments, the cancer is selected from the group consisting of: glioblastoma, melanoma, sarcoma, head and neck cancer, and melanoma.

In some embodiments, the cancer is selected from the group consisting of: glioblastoma, melanoma, sarcoma, and head and neck cancer.

In other embodiments, the cancer is selected from the group consisting of: melanoma, and head and neck cancer.

In some embodiments, the cancer is selected from the group consisting of: melanoma and sarcoma.

In other embodiments, the cancer is selected from the group consisting of: melanoma and glioblastoma.

In some embodiments, the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein and/or the reprogrammed or induced cell as described herein are used as a monotherapy or in combination with other anti-cancer therapeutic(s), such as immunotherapy, such as immune checkpoint blockade inhibitor(s), preferably anti-PD1 , anti-PD-L1 , or anti-CTLA4 therapeutic(s).

In other embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell and the other anti-cancer therapeutic(s) are administered simultaneously, sequentially or separately.

In some embodiments of the uses of the present invention, the cancer is resistant to immune checkpoint blockade inhibition therapy.

In preferred embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are administered intratumorally or systemically, such as intravenously, intraperitoneally, or subcutaneously.

In some embodiments of the uses of the present invention, the use further comprises measuring the number of intratumoral CD8+ T cells, CD4+ T cells, NK cells and/or B cells.

The skilled person will know that tumor cells often fail to activate T cells due to downregulation of antigen presentation pathways, the creation of an immunosuppressive tumor microenvironment (TME), and the absence or dysfunction of professional antigen presenting cells, such as dendritic cells (DCs). The present invention further provides solutions to these challenges.

In preferred embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell increase the number of intratumoral lymphocytes, such as CD8+ T cells, CD4+ T cells, NK cells and/or B cells, preferably the number of intratumoral CD8+ T cells. In preferred embodiments, the intratumoral CD8+ T cells have a more activated effector phenotype (T-bet+PD-1-) and less terminally exhausted (T-bet-PD-1+) phenotype.

In other preferred embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein increase tumor infiltrating lymphocyte (TIL) B cell populations, such as lgM⁺ B cells (CD19+ B220+ lgM+), activated B cells (CD19+ B220+ CD95+ GL7+), plasma cells (CD45+ CD19- CD138+), and/or follicular dendritic cells (DCs) (CD45+ CD19- CD23+).

This may be measured following administration to the subject of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell as described herein. Measurement of the different TIL populations may be performed by any suitable approach known in the art.

In some embodiments, the measurement is performed by tumor immunophenotyping such as described in the Examples herein.

In some preferred embodiments the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein activate the production of circulating tumor-specific IgM, lgG1 , and/or IgA antibodies. Tumor-specific antibody production may be measured by methods known in the art. In some embodiments, tumor-specific antibody production is measured by tumor-specific antibody binding assays, for example as described in the Examples herein, such as using serum from subjects treated with the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for as described herein.

The binding readout may also be measured by methods known in the art. In some preferred embodiments, the readout for antibody production is fluorescence.

In some embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell increase the number of effector and/or central memory T cells, such as CD44+CD62L+ and CD44+CD62L- T cells, respectively.

In other embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell increase the proliferation of CD8+ T cells and/or CD4+ T cells.

In further embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell increase the number of TCF-1+CD8+ T cells and/or the number of TCF-1+CD4+ T cells. In some embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell reduces T cell exhaustion, preferably intratumoral T cell exhaustion.

In some embodiments of the uses of the present invention, the use further comprises measuring the presence of tertiary lymphoid structures (TLS), such as intratumoral tertiary lymphoid structures (TLS), such as tertiary lymphoid structures (TLS) in the tumor parenchyma. In preferred embodiments, the presence of tertiary lymphoid structures (TLS) is performed based on podoplanin expression, such as podoplanin immunostaining.

In other embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell leads to the formation of tertiary lymphoid structures (TLS), or increases the formation of tertiary lymphoid structures (TLS).

In preferred embodiments, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell leads to at least one effect selected from the group consisting of: delay of tumor growth, inhibition of turn or growth, tumor regression, reduced metastasis, abscopal effect, increase in overall survival, complete response, partial response.

In preferred embodiments, the one or more vectors are adenoviral Ad5 vectors comprising the constructs as described herein and abscopal effect is achieved.

In other embodiments, the increase of the at least one effects disclosed herein are observed after at least 1, such as at least 2, such as at least 3, such as at least 4 administrations of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell.

In preferred embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, the methods as described herein, or the uses as described herein, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are administered to an individual in need thereof 1 time, 2 times, 3 times, or 4 times.

In other embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, the methods as described herein, or the uses as described herein, the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are administered to an individual in need thereof at least 3 times, such as at least 4 times, such as at least 5 times.

In some embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, the methods as described herein, or the uses as described herein, immune checkpoint inhibitors are further administered to the individual in need thereof. In preferred embodiments, the immune checkpoint inhibitors comprise anti-PD1 or anti- CTLA-4 antibodies.

In other preferred embodiments, the immune checkpoint inhibitors are administered to the individual in need thereof at least 3 times, such as at least 4 times, such as at least 5 times.

In some embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, the immune checkpoint inhibitors may be administered in any suitable manner, including simultaneously, sequentially, and/or intercalated.

The present disclosure also supports that it may be beneficial in the medical uses and methods of treatments described herein that a first lead cycle of administrations of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell is performed, followed by a second cycle of “booster” administrations.

Thus in some embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, a first lead cycle of administrations of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell is performed, followed by a second cycle of “booster” administrations.

In preferred embodiments, the lead cycle comprises 3 administrations, and the second cycle booster comprises 2 administrations. In even more preferred embodiments, the lead cycle administrations are performed at days 0, 2 and 4, and the second cycle booster administrations are performed at days 9 and 14, wherein day 0 is the day of the first administration.

In further embodiments, the lead cycle administrations are performed at days 0, 2, 4, and 7, and booster administrations are performed at weeks 2, 3, 4 and 5, and immune checkpoint inhibitors are administered at days 0, 3, and 7, wherein day 0 is the day of the first administration.

In yet other embodiments, the lead cycle administrations are performed at days 0, 2 and 4, the booster administrations are performed at days 9 and 14, and immune checkpoint inhibitors are administered at days 0, 3, and 7, wherein day 0 is the day of the first administration.

In further embodiments, the lead cycle administrations are performed at days 0, 2, 4, and 6, and immune checkpoint inhibitors are administered at days 0, 3, and 6, wherein day 0 is the day of the first administration, as described in Example 21 herein.

In some embodiments of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use as described herein, said use comprises an abscopal effect.

In another aspect, the present invention provides a method of treating cancer, the method comprising administering to an individual in need thereof the one or more constructs as described herein, the one or more vectors as described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell as described herein.

Said method may comprise further steps according to the medical uses as described herein.

A further aspect of the present invention relates to the use of the one or more constructs described herein, the one or more vectors described herein, the cell as described herein, the pharmaceutical composition as described herein, and/or the reprogrammed or induced cell as described herein, for the manufacture of a medicament for the treatment of cancer.

Methods for determining treatment efficacy and/or patient response

Another aspect of the present invention relates to a method for determining efficacy to treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell as described herein, comprising: determining the levels of tumor infiltrating lymphocyte (TIL) B cell populations, such as lgM⁺ B cells (CD19+ B220+ lgM+), activated B cells (CD19+ B220+ CD95+ GL7+), plasma cells (CD45+ CD19- CD138+), and/or follicular dendritic cells (DCs) (CD45+ CD19- CD23+) in a first a biological sample of an individual having received the treatment; comparing the levels of the TILs with the levels of the TILs in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said TILs in the first sample compared to the second sample indicates efficacious treatment.

In preferred embodiments, the biological sample is a tumor biopsy.

In other preferred embodiments, the step of determining is performed by tumor immunophenotyping, such as tumor immunophenotyping as described herein.

Another aspect of the present invention relates to a method for determining efficacy to treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell as described herein, comprising: determining the levels of tumor-specific IgM, lgG1, and/or IgA antibodies in a first a biological sample of an individual having received the treatment; comparing the levels of the tumor-specific IgM, IgG 1 , and/or IgA antibodies with the levels of the tumor-specific IgM, lgG1 , and/or IgA antibodies in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said tumor-specific IgM, lgG1 , and/or IgA antibodies in the first sample compared to the second sample indicates efficacious treatment.

In preferred embodiments, the biological sample is a blood sample or a serum sample. In other preferred embodiments, the step of determining is performed by tumor-specific antibody binding assays, such as tumor-specific antibody binding assays as described herein.

Examples

Example 1. General methods and materials.

Mice

Animal care and experimental procedures were performed in accordance with the Swedish federal regulations after approval from the Swedish Board of Agriculture. B6.129S(C)-Batf3tm1 Kmm/J (BATF3KO, The Jackson Laboratory) and C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT-I, The Jackson Laboratory) mice were bred in-house. C57BL/6J, NOD.Cg-PrkdcSCIDIL2rgtm1Wjl/SzJ (NSG, The Jackson Laboratory) and NOD-PrkdcSCITIL2rgtm1/Rj (NXG, Janvier Labs) females aged 6-8 weeks were purchased from Charles River or Janvier-Labs. Animals were housed in a controlled temperature environment (23±2 °C) and a fixed 12-hour light/dark cycle, having free access to food and water. Mice were age-matched, gender-matched and within the same gender randomly assigned to treatment or control groups in all experiments. Numbers of mice for in vivo experiments were determined based on previous expertise, and power analysis was not performed. Mice were sacrificed by cervical dislocation when endpoints were reached. Investigators were not blinded during experimental procedures or the assessment of outcomes.

Cell culture

Mouse B16-F10, LLC, MC38, embryonic fibroblasts (MEFs) and human A375, A2058, HO1u1, IGR39, MCF7, PK59, SKLMS1 , SKMel5, Ca922, 88MEL, T98G cancer cell lines, cancer-associated fibroblasts (CAF), dermal fibroblasts (HDFs) and embryonic kidney (HEK) 293T were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM GlutaMAX, 1 mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM complete). B16- F10 expressing Ovalbumin (B16-OVA) were maintained in DMEM complete supplemented with 0.4 mg/ml geneticin (Gibco). Mouse Panc02, B2905, MB49, BRAFV600ECOX1/2KO cancer cell lines, mouse CD103+ bone marrow-derived dendritic cells (BM-DC), primary mouse and human T cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 2 mM GlutaMAX, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol and 100 U/ml penicillin and 100 mg/ml streptomycin (RPMI complete). YUMM1.7 melanoma cells were cultured in DMEM/F-12 with 10% (v/v) FBS, 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM/F-12 complete). MDSCs were differentiated from monocytes obtained from PBMCs of healthy donors and cultured in RPMI complete. Human pericytes were cultured in Pericyte medium (ScienCell). Fibroblasts were expanded on tissue-culture plates coated with 0.1 % gelatin. All cells were dissociated from tissue-culture plates using TrypLE Express for 5-10 minutes at 37°C, split at 80% confluency and maintained in a humid environment at 37°C and 5% CO2. Reagents used for cell culture were purchased from Thermo Fisher Scientific, STEMCELL Technologies, and Nordic Biolabs.

Molecular cloning

Polycistronic lentiviral vector expressing the mouse or human transcription factors PU.1 , IRF8 and BATF3 separated by 2A self-cleaving peptide sequences under the control of a constitutive SFFV promoter, followed by IRES2-eGFP was cloned previously (Rosa et al. 2022, Zimmermannova et al. 2023). To generate mCherry expressing vectors, the inventors used the empty backbone pRRL.PPT-SFFV-MCS- IRES2 (SFFV-MCS) (Rosa et al. 2022, Zimmermannova et al. 2023) and inserted the coding sequence for mCherry by infusion cloning downstream the IRES sequence to generate pRRL.PPT-SFFV-MCS-IRES2-mCherry (SFFV-mCherry). To generate a lentiviral polycistronic construct for myeloid reprogramming, the coding sequences of mouse PU.1 and C/EBPa (PC) separated by a T2A sequence were cloned first into the MCS of the pFUW-tetO-MCS vector (Rosa et al. 2022) followed by subcloning of the polycistronic cassette into the MCS of the pRRL.PPT-SFFV-MCS-IRES2-eGFP vector (PC-eGFP). Adenoviral vectors (Ad) and adeno-associated viral (AAV) vectors were cloned and produced at VectorBuilder. Replication-deficient adenoviral vectors pAd5- SFFV-PU.1-P2A-IRF8-T2A-BATF3 (Ad-PIB) and pAd5-SFFV-PU.1-P2A-IRF8-T2A- BATF3-CMV-eGFP (Ad-Pl B-eGFP) with an eGFP sequence under the control of constitutive cytomegalovirus (CMV) promoter were generated. pAd5-CMV-eGFP (Ad- eGFP), pAd5-SFFV-Stuffer (Ad-Stuffer) and pAd5-SFFV-Stuffer-CMV-eGFP (Ad- Stuffer-eGFP) were cloned and used as controls. The stuffer sequence was derived from the genome of E. Coli as a non-coding sequence and designed to have the same base pair length as polycistronic PIB. For replication-deficient AAV vectors, pAAV6- SFFV-PU.1-P2A-IRF8-T2A-BATF3 (AAV-PIB) was cloned. To generate eGFP expressing AAV vectors, the stop codon from BATF3 was removed and the eGFP sequence cloned downstream, separated by a F2A sequence (AAV-PIB-eGFP). pAAV6-CMV-eGFP (AAV-eGFP) was used as control. Sequences were verified by Sanger sequencing.

Viral production

Transfer plasmids encoding PU.1 , IRF8 and BATF3 followed by IRES-eGFP (PIB- eGFP), eGFP, PIB-mCherry, mCherry, mOrange and bicistronic PU.1 and C/EBPD followed by IRES-eGFP (PC-eGFP) were used to produce lentiviral vectors. In experiments using lentiviral vectors for in vitro transduction, lentivirus was produced using the second-generation system as previously described (Zimmermannova et al. 2023, Ferreira et al. 2023). In brief, human embryonic kidney (HEK) 293T cells were seeded in 15 cm plates to reach ~80% confluency and transfected with 7.5 pg packaging plasmid (psPAX2), 2.5 pg VSV-G-encoding envelope plasmid (pMD2), and 10 pg transfer plasmid combined with 60 pl of 1 mg/ml polyethyleneimine (PEI) in Opti- MEM. Virus-containing supernatants were collected after 36, 48, and 72 hours, filtered using 0.45 pm low protein binding cellulose acetate filters and concentrated 100-fold with Lenti-X Concentrator (Takara) before storage at -80°C. Alternatively, viruscontaining supernatants were ultracentrifuged for 90 minutes at 4°C with 25,000 g in a SW 32 Ti Swinging-Bucket Rotor (Beckman Coulter). Lentiviral vector pellets were resuspended overnight in DMEM medium and stored in aliquots at -80°C. Lentiviral titers were quantified with the Lenti-X qRT-PCR titration kit (Takara) following the manufacturer’s protocol.

In experiments using lentiviral vectors for in situ transduction, in vivo grade lentiviral particles were produced at VectorBuilder based on the third-generation system. In brief, HEK 293T cells were transfected with eGFP encoding transfer plasmid, envelope plasmid encoding VSV-G and two packaging plasmids encoding Gag/Pol and Rev. The supernatants were collected, and cell debris removed via centrifugation and filtration. Lentiviral particles were subsequently concentrated using polyethylene glycol (PEG) precipitation and further purified through sucrose cushion ultracentrifugation. Lentiviral titers were determined by quantifying the lentiviral p24 Gag protein using ELISA. Adenoviral (Ad) and adeno-associated viral (AAV) vectors encoding for PI B (Ad-Pl B or Ad5-PIB, Ad5-RGD-PIB, Ad5/F35-PIB, Ad5/3-PIB, AAV-PIB) or PIB and eGFP (Ad- PIB-eGFP, AAV-PIB-eGFP), eGFP (Ad-eGFP, AAV-eGFP) or a non-coding stuffer sequence with or without eGFP (Ad-Stuffer, Ad-Stuffer-eGFP) were produced at VectorBuilder, Vector Biolabs or O.D.260 Inc, using O.D.260 Inc AdenoQuick 2.0 cloning system for Ad5-PIB, Ad5-RGD-PIB, Ad5/F35-PIB, Ad5/3-PIB. Adenoviral vectors were packaged and amplified in HEK 293A cells. In brief, adenoviral plasmids containing PIB, PIB-eGFP, Stuffer or eGFP were first linearized by restriction digestion with Pad. The linearized plasmid DNA was then transfected into HEK 293A expressing the adenovirus gene E1 to produce recombinant adenovirus. Adenoviral particles released into the culture medium were harvested and concentrated using cesium chloride (CsCI) gradient ultracentrifugation and/or chromatography. The viral titer was determined by spectrophotometry (OD260) to quantify the number of viral particles and measured for the number of infective units (IFU) by immunocytochemistry staining for transduced cells via the detection of adenovirus-specific hexon protein.

For AAV production, HEK 293T cells were co-transfected with a helper plasmid encoding adenovirus genes (E4, E2A, and VA), an AAV helper plasmid encoding AAV rep and cap genes, and a transfer plasmid containing PIB-eGFP or eGFP. After incubation, AAV particles were harvested from cell lysates and supernatant and concentrated using polyethylene glycol (PEG) precipitation. Subsequently, AAV particles were further purified and concentrated through CsCI gradient ultracentrifugation. The viral titer was determined by quantitative PCR (qPCR) targeting the inverted terminal repeat sequence of AAV. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were used to determine AAV purity. All viral vectors used for in vivo application successfully passed sterility and mycoplasma testing.

Flow cytometry and Fluorescence-activated cell sorting (FACS)

Surface marker analysis was performed on dissociated cells from in vitro 2D cultures or single cell suspensions of digested tissue from spheroids or tissues. Cells were stained with adequate antibodies diluted in phosphate-buffered saline (PBS) supplemented with 2% FBS (FACS buffer) at 4°C for 20-30 minutes in the presence of 1% mouse or rat serum, for human and mouse cells, respectively, to block unspecific binding. Tetramer staining was performed at room temperature for 30 minutes before surface marker staining and cell fixation using 4% paraformaldehyde (PFA, Thermo Fisher Scientific) for 20 minutes at 4°C without permeabilization. Intracellular staining for cytokines or proliferation marker Ki67 was performed using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) following the manufacturer's recommendation. Intranuclear transcription factor staining was performed using the True-Nuclear Transcription Factor Buffer Set (Biolegend) following the manufacturer's recommendation. For flow cytometry analysis requiring cell fixation, cells were stained with fixable viability dye (FVD) 450 or 520 (Thermo Fisher Scientific) before surface marker staining and fixation. For analysis without cell fixation, dead cells were excluded by addition of 4',6-diamidino-2-phenylindole (DAPI) or 7-aminoactinomycin D (7-AAD) to the cell suspension after surface marker staining and before acquisition. Flow cytometry analysis was performed on LSR Fortessa, LSR Fortessa X20, FACSymphony A1 and Beckman Coulter Life Sciences CytoFlex Benchtop flow cytometers. FACS-sorting was performed on a BD FACS Aria III or on a FACSymphony S6 sorter, using a 100 pm nozzle. FACS data were analyzed using FlowJo v.10.0.7 (FlowJoLLC). Gates were determined according to fluorescence minus one (FMO) controls.

In vitro transduction and reprogramming of tumor cells

In vitro reprogramming mediated by lentiviral vectors was performed as previously described (Zimmermannova et al. 2023, Ferreira et al. 2023). In brief, 0.5-1x10⁶ cells were plated per tissue culture 6-well or 10-cm plate and incubated overnight with 5.5x10⁷ and 5.0x10^s genomic copies (GC) per cell in the presence of 8 pg/ml polybrene. Adenoviral and adeno-associated viral vectors were used at a multiplicity of infection of 5,000 IFU per cell for Ad vectors and 250,000 GC per cell for AAV vectors in tissue culture 12-well, 6-well or 10-cm plates. Transduction was performed in 5 ml in a 10-cm plate, 1 ml per well in a 6-well plate, or 0.5 ml per well in a 12-well plate. After 16h, virus-containing medium was replaced with fresh medium and cells were maintained until day 9 in culture with regular medium changes every 2-3 days and splitted 1 :6 when 80% confluency was reached. Transduction efficiency was measured by flow cytometry using eGFP expression. cDC1 reprogramming efficiency was measured by flow cytometry analysis of CD45 and MHC-II/H LA-DR expression within live or live eGFP+. Macrophage reprogramming efficiency was measured by CD45 and CD11b expression within live eGFP+ cells. In addition, the expression of MHC-I/HLA- ABC, the co-stimulatory molecule CD40 and cDC1 markers XCR1 , CLEC9A and CD226 was quantified by flow cytometry. Quantification of MHC-I/HLA-ABC surface molecules per cell was performed using the PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences) following manufacturer’s instructions.

Spheroid formation

Human cancer cell line-derived spheroids were formed using the forced-floating method. Each well of an ultra-low-attachment U-bottom 96-well plate (Perkin-Elmer) was seeded with 2x10⁴ cells in 200 pL of respective medium supplemented with 2.5% Matrigel (Corning) followed by centrifugation at 1 ,000 g for 10 minutes to force cell aggregation. The plates were incubated at 37°C for 96 hours after which tumor spheroid formation can be observed. To form the multi-component heterotypic spheroids, eGFP-transduced T98G (T98G-eGFP+) cells and cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs) or pericytes were counted, combined at desired ratios, and 2x104 cells were seeded as described above. 100 pL of medium containing 2.5% Matrigel was replaced every 3-5 days of the experiment. 50% of media was carefully replaced to avoid organoid disruption. For immunohistochemistry, confocal imaging, ATP release assay, and co-cultures with PBMC, the spheroids were aggregated from a suspension of 300 cancer cells and 1 ,000 CAF over a period of 3 days without the addition of Matrigel. Alternatively, for human cancer cell line-derived spheroid co-cultures with PBMCs, spheroids were formed with MACS-enriched CD45+ T98G cells at day 7 of reprogramming and mixed with CAFs before addition of PBMCs. For formation of patient-derived cancer spheroids, cancer cells were resuspended in 200 pl of DM EM or PneumaCult complete medium containing 2.5% Matrigel and dispensed to single wells of a 96 Thermo Scientific Nunclon Sphe 3D culture system plate. When indicated, patient-derived cancer spheroids were formed with CAFs. Prior to co-culturing CAFs with cancer cells, CAFs were transduced with the lentiviral vector SFFV-mOrange to separate CAFs based on fluorescent mOrange expression from cancer cells. Spheroids were kept in these conditions for a maximum of 3 days before viral transduction.

Tumor establishment

To establish tumors, cancer cells were harvested with TrypLE Express, live cells counted by Trypan blue staining using an automated hemocytometer and injected subcutaneously into the right flanks of recipient mice in 100 pl of ice-cold PBS. Before injection, mice were anesthetized by an intraperitoneal injection of ketamine (135 mg/kg) and xylazine (3 mg/kg). For tumor growth and survival experiments, 1x10⁵ B16- F10, YUMM1.7 or 1x10⁶ B2905 in C57BL/6J mice, 1x10⁵ YUMM1.7 in NSG mice, or 1x105 BRAF^V600ECOX1/2^K° cells in BATF3^KO mice were used. In bilateral tumor settings, 2x105 B16-OVA, B16-F10 or YUMM1.7 were injected subcutaneously into the right flank and 1x105 B16-OVA, B16-F10 or YUMM1.7 into the left flank. LLC tumors were formed by subcutaneous injection of 1x10⁶ cells into the upper right neck area. For immunophenotyping and immunofluorescence analysis of tumors established with mixtures of in vitro transduced and parental cells, a total of 1x10⁶ B16-F10 or YUMM1.7 cells were injected in C57BL/6J mice. For establishing xenograft models, 5x10⁵ of human A375, A2058, T98G in NSG or A2058 and SKLMS1 cell lines were injected into NXG mice. C57BL/6J, NSG and NXG mice were 6-12-week-old age-matched females and BATF3KO mice were males and females 6-12 weeks old. Tumor volumes were monitored with a digital caliper and calculated using the formula V = L*W*H/2. Survival was determined by predefined endpoints such as tumor size reaching 1500 mm3, tumor ulceration, or signs of animal suffering. Animals were randomized for tumor establishment and again before treatment.

In vivo reprogramming of tumor cells

To evaluate the immunogenicity of in vivo reprogrammed cells in syngeneic mouse melanoma models, the inventors transduced cancer cells in vitro with lentiviral (PIB, PC) or adenoviral particles (Ad-PIB), and 16 hours post-transduction mixed with untransduced parental cancer cells in defined ratios and injected subcutaneously into the right flank of mice. Unless stated otherwise, cells were mixed at a 1 :1 ratio of transduced and untransduced parental cancer cells. As controls, empty viral vectors (lentivirus control: eGFP, adenovirus control: Ad-Stuffer) were used. Transduction with lentiviral vectors was performed in the presence of polybrene (8 pg/ml, Sigma-Aldrich). The MOI used for transduction and induction of reprogramming by lentivirus ranged between 5.5x10⁷ and 5.0x10⁸ GC per cell. Cell mixtures were also kept in vitro to estimate the percentages of transduced cells by eGFP expression at day 3 and reprogramming efficiency by CD45 and MHC-II expression at day 9 by flow cytometry. To establish a dose-response relationship between the amount of transduced and reprogrammed cancer cells with induced antitumor immunity, transduced cells were serially diluted with increasing amounts of parental cancer cells (1 :1 , 1 :2, 1 :4, 1 :10 and 1 :100 ratio) before subcutaneous injection into mice. For adenoviral-mediated reprogramming in vivo, cells were transduced with non-eGFP encoding vectors (Ad- PIB, control: Ad-Stuffer) at an MOI of 2,500 infective units (IFU) per cell. To characterize the in vivo reprogramming efficiency of human cancer cells, the human cancer cell lines T98G, A375, A2058 were used in NSG mice. Cells were transduced with lentiviral vectors (PIB-eGFP, control: eGFP) mixed with untransduced cells and injected subcutaneously 16 hours post transduction and kept in vitro for phenotypic profiling by flow cytometry. At days 3, 5 and 9 post tumor establishment, tumors were isolated and dissociated into single cell suspensions for flow cytometry analysis for tumor or melanoma markers (CD44, MCSP), reprogramming markers (CD45, HLA- DR), antigen presentation complex (HLA-ABC), co-stimulatory molecule (CD40) and cDC1 markers (XCR1 , CLEC9A, CD226).

Immune checkpoint blockade treatment

For single or combinatorial treatment with ICB, mice received 200 pg of anti-PD-1 (clone RMP1-14, BioXCell) and/or 200 pg of anti-CTLA-4 (clone 9H10, BioXCell) or rat 200 pg lgG2a (clone 2A3, BioXCell) and lgG2b (clone LTF-2, BioXCell) isotype control antibodies diluted in 100 pl PBS intraperitoneally at days 7, 10, and 13 after tumor establishment.

Magnetic-activated cell sorting (MACS) enrichment

To purify in vitro reprogrammed mouse cancer cells by magnetic-activated cell sorting (MACS, Miltenyi) with high yields, the inventors followed a protocol as previously described (Ferreira et al. 2023). Briefly, cells were resuspended in cold FACS buffer to reach a concentration of 10⁷ cells/ml and incubated with rat serum for 15 minutes, followed by 5 minutes incubation with biotinylated antibodies. To purify cDC1-like cancer cells, biotinylated CD45 and MHC-II antibodies were used. To purify macrophage-like cancer cells, biotinylated CD45 and CD11 b antibodies were used. Cells were washed twice with FACS buffer before incubation with magnetic anti-biotin microbeads for 15 minutes. All incubations were performed on ice and labelled cells were purified using LS columns (Miltenyi) according to the manufacturer's recommendations. To purify naive ovalbumin-specific CD8+ T cells from spleens of OT-I mice, spleens were isolated and homogenized by plunging against a 40 pm cell strainer. Red blood cells were lysed with Pharm lyse lysing buffer (BD Biosciences) for 8 minutes protected from light at room temperature and naive CD8+ T cells were purified by MACS using the naive mouse CD8+ T cell isolation kit (Miltenyi Biotec). To purify human HLA-A2+ CD8+ T cells, peripheral blood mononuclear cells (PBMCs) from HLA-A2+ donors were centrifuged at 350g for 5 minutes at room temperature, resuspended in FACS buffer and CD8+ T cells were isolated using the human CD8+ T cell isolation kit (M iltenyi) according to manufacturer’s recommendations.

T cell re-stimulation of mouse peripheral blood

Peripheral CD8+ and CD4+ T cells from treated and control animals were isolated from peripheral blood by tail vein puncture and collected into K2-EDTA coated microvette tubes (Sarstedt). Erythrocytes were lysed using Pharm lyse lysing buffer (BD Bioscience). After lysis, live cells were resuspended in RPMI complete medium and plated in 96-well U-bottom plates at 2x10⁵ cells per well for antigen-agnostic or antigenspecific T cell re-stimulation.

For antigen-agnostic T cell re-stimulation, 1x105 cancer cells were plated per well and stimulated with 100 ng/ml mouse IFNy (Miltenyi) 24 hours before start of the co-culture with peripheral blood T cells. Before addition of T cells, medium supplemented with IFNy was removed, cancer cells were washed with PBS and T cells were added in RPMI complete medium. After 1 hour, brefeldin A (BD Biosciences) was added to block secretion of intracellular proteins and the co-culture continued for 5 hours. For antigenspecific T cell re-stimulation, 1x10⁴ CD103+ BM-DCs were plated into 96-well U-bottom plates with 10 pg/ml of tumor antigen-derived peptides TRP-2 (MBL), PM EL (AnaSpec) and p15E (MBL) 24 hours before addition of T cells. BM-DC and T cell co-cultures were performed for 16 hours, then brefeldin A was added to the medium and cells were incubated for additional 5 hours. Intracellular production of IFNy in T cells was analyzed by flow cytometry following cell fixation and intracellular antibody staining.

In vivo delivery of viral vectors

To deliver viral vectors to tumors in situ, LV, Ad, AAV vectors were diluted in ice-cold PBS to reach a final volume of 30 pl and intratumorally injected when the size of tumors reached 30-90 mm3. Tumors that did not reach the required sizes were excluded from the experiment. To quantify in vivo transduction efficiency in B16 tumors, eGFP-encoding vectors were administered at day 7 and 9 post tumor establishment. 4x10⁵, 4x10⁶ and 4x10⁷ GCs of LV-eGFP, 10⁸, 10⁹ and 10¹° viral particles (VPs) of Ad-eGFP and 8x10⁸, 8x10⁹ and 8x10¹° GCs of AAV-eGFP were administered per injection. At day 12, tumor tissue was isolated and dissociated into single cell suspensions for flow cytometry analysis of transduction efficiency through quantification of eGFP+ cells within live CD44+CD45- cells. To assess in situ reprogramming efficiency in human xenograft models or efficacy in the B16 model combined with ICB treatment, 1010 VPs of Ad-PIB-eGFP or Ad-Stuffer-eGFP were injected intratumorally at day 7, 9, 11 and 13 post tumor establishment. In human xenograft models vectors encoded also eGFP (Ad-PIB-eGFP, Ad-Stuffer-eGFP). At day 16 after human xenograft establishment in NXG mice, tumors were isolated, dissociated, and reprogramming efficiency quantified by flow cytometry.

Statistical analysis

All statistical analyses were performed using GraphPad Prism or R software. Data was subjected to a normality test before using ANOVA, two-way ANOVA, Kruskal-Wallis or Mann-Whitney test and t-test. Statistical significance of two groups was determined using an unpaired two-tailed Mann-Whitney test or t-test. Group comparisons were performed using ANOVA and corrected by Dunn's or Tukey's multiple comparison test. To estimate statistically significant differences in the survival in multiple groups the logrank Mantel-Cox test was used. Unless stated otherwise in the figure legends, data are shown as mean ± SD and n represents the total number of animals in in vivo experiments or biological replicates in in vitro experiments. Randomization was performed using the Microsoft Office Excel function (=RANDBETWEEN). Sample sizes were based on previous experience. Significance was considered with *p < 0.05; **p < 0.01; ***p < 0.001 ; ****p < 0.0001.

Example 2. In vivo cDC1 reprograming induces antitumor immunity and allows higher in vivo efficacy when compared to in vivo myeloid reprogramming.

Background

The inventors previously identified the combination of transcription factors composed by PU.1 , IRF8, and BATF3 (PIB) as sufficient to reprogram fibroblasts or tumor cells into cDC1-like cells in vitro endowed with the three signals required to activate T cells, including antigen presentation on MHC class I and II, co-stimulatory molecule expression and chemokine/cytokine secretion (Rosa et al. 2018, Rosa et al. 2022, Zimmermannova et al. 2023). Here, the inventors hypothesized that PIB mediate the reprogramming of tumor cells into immunogenic cDC1-like cells entirely in vivo within the TME and benchmarked antitumor efficacy of in vivo cDC1 reprogramming mediated by PIB benchmarked to myeloid reprogramming mediated by PU.1 and CEBP/a. Methods

Magnetic-activated cell sorting (MACS) enrichment

To purify in vitro reprogrammed mouse cancer cells by magnetic-activated cell sorting (MACS, Miltenyi) with high yields, a protocol was followed as previously described (Ferreira et al. 2023). Briefly, cells were resuspended in cold FACS buffer to reach a concentration of 107 cells/ml and incubated with rat serum for 15 minutes, followed by 5 minutes incubation with biotinylated antibodies. To purify cDC1-like cancer cells, biotinylated CD45 and MHC-II antibodies were used. To purify macrophage-like cancer cells, biotinylated CD45 and CD11 b antibodies were used. Cells were washed twice with FACS buffer before incubation with magnetic anti-biotin microbeads for 15 minutes. All incubations were performed on ice and labelled cells were purified using LS columns (Miltenyi) according to the manufacturer's recommendations. To purify naive ovalbumin-specific CD8+ T cells from spleens of OT-I mice, spleens were isolated and homogenized by plunging against a 40 pm cell strainer. Red blood cells were lysed with Pharm lyse lysing buffer (BD Biosciences) for 8 minutes protected from light at room temperature and naive CD8+ T cells were purified by MACS using the naive mouse CD8+ T cell isolation kit (Miltenyi Biotec).

Generation of bone marrow-derived CD103+ dendritic cells

Mouse CD103+ bone marrow-derived dendritic cells (BM-DCs) were generated as previously described (53). In brief, bone marrow (BM) cells were harvested from long bones of the leg (tibias and femurs) by crushing. Cells were then harvested in PBS supplemented with 2% FBS, filtered with a 40 pm cell strainer and plated in Petri dishes at a density of 1.5x10⁶ cells per mL of RPMI complete medium supplemented with 5 ng/ml GM-CSF (Miltenyi) and 200 ng/ml Fltl3L (Miltenyi) for 16 days (Mayer et al. 2014).

Cross-presentation assay

Naive ovalbumin-specific CD8+ T cells from spleens of OT-I mice were enriched using a naive mouse CD8+ T cell isolation kit (Miltenyi Biotec). Enriched CD8+ T cells were labeled with CellTrace Violet (CTV, Thermo Fisher Scientific) according to manufacturer’s protocol. B16 cells were transduced with lentiviral particles encoding PIB-eGFP or PC-eGFP and reprogrammed for 9 days into cDC1-like or macrophagelike cancer cells, respectively. MACS-purified reprogrammed cancer cells, eGFP- transduced cancer cells, and CD103+ BM-DCs were incubated overnight at 37°C with ovalbumin protein (100 pg/ml) in the presence of P(I:C) (10 pg/ml) and extensively washed. Then, 1x10⁴ cells were incubated with 1x10⁵ naive CTV-labeled OT-I CD8+ T cells in 96-well U-bottom plate. After 3 days of co-culture, T cells were collected, stained, and analyzed by flow cytometry. T cell proliferation was determined by dilution of CTV staining and upregulation of CD44 expression. The threshold for data plotting was fixed at 100 events within live CD8+ T cell gating.

Results

To test in vivo cDC1 reprogramming the inventors subcutaneously implanted a mixture of 88% PIB-eGFP-transduced B16 cells and 12% untransduced parental cells, or mixtures of eGFP-transduced and parental cells as a control, 16 hours after transduction (Figure 1A). This strategy allowed to separate delivery of the transcription factors from the in vivo reprogramming process. The inventors observed complete responses (CR) in 30% of animals and delayed tumor growth in the other animals, thereby extending median survival (MS, 43 vs. 19 days, p<0.0001). When combined with anti-PD-1 and anti-CTLA-4, tumor regression was observed in all animals. To dissect whether cDCT functional properties are critical for the observed potent antitumor immunity the inventors compared cDC1 reprogramming with myeloid reprogramming mediated by PU.1 and C/EBPa (PB) to induce macrophage-like cells (Linde et al. 2023) (Fig. 1A). In vivo, cDC1 reprogramming extended MS when compared to macrophage reprogramming (43 vs. 29.5 days, p<0.0001), especially when combined with ICB (p=0.0003), which resulted in 100% CRs (Figure 1A). This effect is consistent with the selective induction of high levels of MHC-I and MHC-II (Fig. 1B) and cross-presentation capacity by PIB (Fig. 1C).

Conclusion

These data suggests that in vivo cDC1 reprogramming induces effective antitumor immunity as monotherapy and in combination with immune checkpoint blockade therapy, and highlights the superior in vivo efficacy induced by cDC1 reprogramming mediated by PU.1, IRF8 and BATF3 when compared to myeloid reprogramming mediated by PU.1 , and C/EBPa. Example 3. In vivo cDC1 reprogramming induces systemic and durable antitumor immunity.

Background

The efficacy of in vivo cDC1 reprogramming was investigated as monotherapy or in combination with either anti-PD-1 or anti-CTLA-4 using B16, B2905 and the additional melanoma model YUMM1.7, which is resistant to ICB and also depends on cDC1 availability (Salmon et al. 2016, Perez-Guijarro et al. 2020, Ghislat et al. 2021)

Results

A 1 :1 mixture of transduced and parental melanoma cells was implanted (Fig. 2A) and it was observed that monotherapy induce complete tumor regressions in YUMM1 .7 (100% CR), B2905 (80% CR), and extended MS in B16 challenged animals from 17 to 31 days (Fig. 2B). cDC1 reprogramming synergized with anti-PD-1 or anti-CTLA-4 treatment leading to increased CRs in B16 and B2905, which also resulted in expansion of tumor antigen-specific IFNy+CD8+ and IFNy+CD4+ T cells in peripheral blood (Fig. 2C). To assess whether antitumor immunity requires endogenous cDC1, the inventors used the immunogenic BRAF^V600ECOX1/2^K° melanoma model which grows in BATF3KO mice due to the lack of endogenous cDC1 (Meiser et al. 2023). Increased MS (96.5 vs. 19.5 days, p<0.001) (Fig. 2A), synergy with anti-PD-1 treatment (50% vs. 100% CR), and concomitant expansion of tumor antigen-specific T cells (Figure 2B) was observed. To address whether immune memory was induced the inventors re-challenged survivor WT or BATF3KO animals that showed complete tumor regressions. While naive mice developed tumors, survivor animals remained tumor- free (100% YUMM1.7; 66% BRAFV600ECOX1/2KO) (Fig. 2C). The inventors then asked whether combination with ICB is required for systemic antitumor immunity and observed abscopal effects with monotherapy or combined with anti-PD-1 or anti-CTLA- 4 (Fig. 2D)

Conclusion

These findings highlight that in vivo cDC1 reprogramming mediated by PU.1 , IRF8 and BATF3 (i) is sufficient to elicit antitumor immunity, (ii) protects from tumor growth of distal non-treated tumors, (iii) induces immunological memory and protects from tumor growth after re-challenge, and (iv) the effects are independent of endogenous cDC1s. Example 4. In vivo cDC1 reprogramming remodels the tumor microenvironment.

Background

Given the ability of cDC1 to shape the TME (Barry et al. 2018, Cohen et al. 2022), the inventors addressed the impact of cDC1 reprogramming on tumor morphology and immune composition by immunofluorescence and flow cytometry.

Methods

Immunohistochemistry and immunofluorescence

For mouse tumor immunofluorescence images, tumors were excised at day 9 post tumor establishment with a 1 :1 mixture of eGFP- or PIB-eGFP-transduced and untransduced parental cells, fixed in 10% formalin for 24 hours, dehydrated for 72 hours by incubation with increasing ethanol concentrations (70%, 95% and absolute ethanol), and embedded in paraffin after xylene treatment. 5 pm thick tissue sections were cut and mounted onto BOND Plus slides (Leica). The paraffin-embedded sections were de-paraffinized and antigen retrieval was performed using a steamer at 99°C in TRIS/EDTA buffer (Biotechne) for 15 minutes. After antigen-retrieval, sections were blocked for 1 hour and antibody staining was performed in a humidified chamber for 2 hours with fluorophore-conjugated antibodies (Novus biologicals) using anti-eGFP- AF594 (1:50), anti-CD45-AF647 (1 :100) and nucleic staining with Syto 13 (50 pM) (Thermo Fisher Scientific) at room temperature. Fluorescence images were acquired using the scanner of GeoMx (Nanostring).

For immunohistochemistry analysis of spheroids, tissues were fixed with 4% PFA and embedded in 1.7% agarose gel. Paraffin embedding, cutting under microscope control, and antibody staining with Hematoxylin and Eosin, Ki67, vimentin, FAP (R&D Systems), EGFR (DBS), and cytokeratin (Dako) was performed at Sophistolab AG. Image acquisition was performed with Leica DMi8 microscope (Leica) using a DMC4500 Camera with a 40x objective.

Immune cell depletion

For depletion of CD8+ T cells, CD4+ T cells or NK cells, mice were injected intraperitoneally with 100 pL of depleting anti-CD8a (clone 53-6.7, BioXCell), anti-CD4 (clone GK1.5, BioXCell), anti-NK1.1 (clone PK136, BioXCell) or rat isotype control lgG2a (clone 2A3, BioXCell) and lgG2b (clone LTF-2, BioXCell) antibodies (400 pg/mouse). Antibodies were injected two days before, the same day, and two days after tumor establishment. Depleting antibody injections were continuously repeated every 3-4 days. Depletion was confirmed by flow cytometry analysis of NK cells, CD8+ and CD4+ T cells from peripheral blood at the same day of tumor establishment.

Results

At day 9 after initiation of in vivo reprogramming, the inventors observed the formation of tertiary lymphoid structures (TLS) in the parenchyma of tumors (Fig. 3A), which contained a B cell and CD4+ T cell zone and a spatially segregated CD8+ T cell zone that were juxtaposed with TLS-specific podoplanin+ stromal cells (Fig. 3A, B). At day 21 , B cell percentages increased by 24.4-fold, NK cells by 2.2-fold, and CD4⁺ T cells by 2.4-fold within PIB-treated tumors (Fig. 3C). Although the percentages of CD8⁺ T cells were similar between treated and untreated tumors, a substantial decrease (4- and 8- fold) of both exhausted PD-1⁺CD8⁺ and PD-1⁺CD4⁺ T cells, respectively was found (Fig. 3D). Conversely, the inventors observed a 4.3-fold increase of central memory CD62L⁺CD44⁺CD8⁺ T cells, that are critical for long-term memory (Micevic et al. 2023), and effector CD44⁺CD4⁺ T cells (Fig. 3E). In agreement with the reduction in exhaustion, a 10.5- and 1.5-fold decrease in regulatory CD8⁺ and CD4⁺ T cell (Treg) populations was observed, indicating that in vivo cDC1 reprogramming within tumors induces a shift towards pro-inflammatory T cell populations (Fig. 3F). Next, the inventors depleted CD8⁺ T cells, CD4⁺ T cells and NK cells with antibodies in WT mice. Surprisingly, it was observed that depletion of CD4⁺ T cells abolished tumor immune control, highlighting the critical role of CD4⁺ T cells in PIB-mediated antitumor immunity (Fig. 3G). While NK cell depletion did not show an impact in tumor growth, CD8⁺ T cell depletion reduced tumor control at later time points in 40% of animals.

Conclusion

Our data show that in vivo cDC1 reprogramming remodels the TME, reduces exhausted and regulatory populations and increases the infiltration of memory and stem-like T cells.

Example 5. In vivo cDC1 reprogramming induces polyclonal cytotoxic and memory T cell responses

Background To characterize T cell responses elicited by in vivo cDC1 reprogramming, the inventors profiled T cells from tumors, tdLN and peripheral blood using single cell RNA- sequencing (scRNA-seq) with T cell receptor (TCR) enrichment (Fig. 4A).

Methods

Single cell RNA sequencing with TCR enrichment of T cells 5' scRNA-seq with TCR enrichment was performed on FACS-sorted CD45⁺CD3⁺ T cells isolated from tumors, tdLN and peripheral blood of animals 21 days after tumor establishment with subcutaneous injection of PIB-eGFP or eGFP-transduced YUMM1.7 cells mixed at a 1 :1 ratio with untransduced parental cells. Tumors were processed into single cell suspensions and tdLN were mechanically dissociated with a plunger against a 50 pm cell strainer and collected in FACS buffer for staining. Blood samples were collected into K2-EDTA coated microvette tubes (Sarstedt) and further processed to remove erythrocytes through red blood cell lysis using BD Pharm Lyse lysing buffer (BD Bioscience). Single cell suspensions were pooled from 5 animals per treatment group and stained with anti-CD45 and anti-CD3 antibodies. 3000-10,000 T cells were FACS-sorted, resuspended in PBS containing 0.04% bovine serum albumin (BSA, STEMCELL Technologies) and loaded on a 10x Chromium (10x Genomics) without multiplexing.

Analysis of single cell RNA sequencing with TCR enrichment of T cells Single cell RNA-seq libraries were prepared using the Chromium Single Cell V(D)J Reagent Kit (10x Genomics). Indexed sequencing libraries were quantified with a High Sensitivity DNA analysis kit (Agilent) and Agilent Bioanalyzer. Indexed libraries were pooled in an equimolar ratio and sequenced with an Illumina NextSeq 500 platform. 10x Genomics cellranger Single Cell Software v7.1.0 was used for demultiplexing, alignment (mm10), filtering, UMI counting, single cell 5' end gene counting, TCR assembly, annotation of paired VDJ and performing quality control using the manufacturer’s parameters. The sparse expression matrix generated by the cellranger analysis pipeline was used as input to the Seurat library v4.3.0, and cells and genes passing quality control thresholds were included according to the following criteria: 1) number of genes detected in each single cell greater than 200 and lower than 5,000; 2) percentage of counts in mitochondrial genes less than 7.5%. Data was normalized using "LogNormalize" with the scale factor of 10,000 and identified 2,500 variable features. The first 30 principal components were used for subsequent UMAP and clustering analyses. Next, the data was segregated into CD4 and CD8 dataset, according to gene expression of Cd4 and Cd8a. Each data set was reanalyzed independently using 2,000 variable features and 15 principal components for UMAP and clustering analysis. The inventors identified 9 clusters for CD8+ and 12 clusters for CD4+ T cell datasets. For differential expression analysis between cell types, FindAIIMarkers function with Wilcox Rank Sum test was used with the following parameters: logfc.threshold = 0.25, min. pct = 0.25. In addition, scaling was performed and resulting genes were visualized using DoHeatmap function and used for annotation of cell types. Additionally, differential expression analysis was performed between differently treated cells for each cluster using the parameters defined above. To order cells on pseudotime, Monocle 3 library v1.3.1 was used. The data was separated by treatment and run on corresponding UMAP with default settings except for the following parameter: use_partition=FALSE that assumes all cells in the dataset descend from a common transcriptional ancestor. The root of the trajectory was selected automatically among early activated cells for CD4 and CD8 cells. The VDJ data generated by the cellranger analysis pipeline was used as input to scRepertoire library v1.8.0 and matched with corresponding Seurat objects. Any chain without values and multi chains were removed by selecting the 2 corresponding chains with the highest expression for a single cell barcode.

Results

The inventors segregated single cells expressing either CD8a or CD4 and performed cluster annotation. 9 CD8⁺ T cell clusters were identified and increased frequencies of intratumoral effector and effector memory cells were observed in PIB-treated tumors, accompanied by a reduction in exhausted and terminally exhausted subsets (Fig. 4B). The frequencies of effector and stem-like CD8⁺ T cells were amplified when in vivo reprogramming was combined with anti-PD-1 treatment. The inventors also identified 11 CD4⁺ T cell clusters (Fig. 4B). In tumors treated with in vivo reprogramming, a large cluster of cytotoxic CD4⁺ T cells was observed that has recently been described to harbor the capacity to directly eradicate melanoma cells (Bawden et al. 2024). Interestingly, when combined with anti-PD-1, CD4⁺ T cells shift towards T helper (Th) precursors, type 1 T helper (Th1), and stem-like fates indicating either differential kinetics or differential priming of these populations. Decreased frequencies of exhausted and regulatory CD4⁺ T cells were also observed. To address if polyclonal expansion was induced, the inventors analysed full-length a.p TCR sequences obtained from single T cells with more than 1 cell per clonotype. First, similar polyclonal expansion of CD8⁺ T cells was observed in tumors treated with in vivo reprogramming and control tumors (118 and 112 unique clones) (Fig. 4C). Interestingly, the expansion of 160 unique CD4⁺ T cell clones was observed while only 57 and 92 were found in control tumors and tumors treated with anti-PD-1. In contrast, the combination with anti-PD-1 treatment reduced CD4⁺ T cell clonal expansion (53 unique clones).

Conclusion

These results further underscore the importance of polyclonal CD4+ T cells for tumor control in line with immune depletion experiments demonstrating the requirement of CD4+ T cells for antitumor immunity induced by cDC1 reprogramming.

Example 6. cDC1 reprogramming progresses in vivo in the context of human tumors

Background

The signals from the environment impact the reprogramming process (Zhou et al. 2008, Qian et al. 2012), and thus, the inventors assessed whether human cancer cells acquire an immunogenic cDC1 phenotype in vivo.

Results

Human melanoma (A375 and A2058) and glioblastoma (T98G) tumors were established in NSG mice using cells transduced in vitro and phenotypic characterization of the in vivo reprogramming process was performed by flow cytometry. At day 9, the inventors detected reprogrammed CD45⁺HLA-DR⁺ T98G (75.36±11.84%), A375 (69.93±11.45%) and A2058 (76.97±7.24%) cells, as well as partially reprogrammed cells expressing either CD45 or HLA-DR (Fig. 5A, B). A gradual increase in the percentage of reprogrammed cells was observed from days 3 to 9, showing not only the initiation but also progression of the reprogramming process in vivo as observed in vitro (Zimmermannova et al. 2023). As a measure of reprogramming fidelity, the expression of the cDC1-specific surface markers XCR1 and CLEC9A was assessed (Fig. 5C). Interestingly, already at day 5 the inventors observed enhanced expression of XCR1 in vivo when compared to in vitro in T98G (8.0±2.5% vs. 1.2±1.1%), A375 (12.1±6.6% vs. 0.4±0.2%) and A2058-derived cells (9.5±6.5% vs. 4.8±3.2%). Regarding the immunogenicity of induced cells in vivo at day 9, a 4-, 2-, and 3-fold higher expression of HLA class I molecules was detected in reprogrammed T98G, A375 and A2058 cells, respectively (Fig. 5D). Moreover, the acquisition of the co-stimulatory molecule CD40 was confirmed, reflecting a mature antigen-presenting phenotype (Fig. 5D).

Conclusion

PI B overexpression in human xenograft models induces a cDC1 phenotype in vivo with enhanced fidelity and immunogenicity.

Example 7. cDC1 reprogramming is independent of the immunosuppressive tumor microenvironment

Background

To further support the feasibility of cDC1 reprogramming in tissues that include the presence of human immunosuppressive cells and soluble mediators of the TME, the inventors profiled cDC1 reprogramming efficiency in human cancer cell-lines derived spheroid models and investigated the impact of immunosuppressive cells and factors in reprogramming efficiency.

Methods

Myeloid-derived suppressor cell differentiation

To prepare MDSCs, PBMCs from peripheral blood of healthy donors were first isolated using Lymphoprep tubes (Thermo Fisher Scientific). Erythrocytes were removed using Pharm Lyse (BD Biosciences) and monocytes were enriched via positive selection using CD14 microbeads (Miltenyi) by magnetic-activated cell sorting (MACS) according to the manufacturer's protocol. CD14⁺ monocytes were cultured in RPMI complete supplemented with 1 ug/ml of TLR2 agonist Pam3CSK4 (1 ug/ml; Invivogen) previously shown to promote MDSC polarization (Wang et al. 2015). Acquisition of an MDSC phenotype was confirmed via flow cytometry quantification of the markers CD163, CD206, and 25F9.

Single cell RNA sequencing of human cancer cells reprogrammed in 2D and 3D scRNA-seq was performed in T98G cells from monolayer culture or isolated from spheroids after the transduction with PIB-eGFP or control vector eGFP. At day 3, 7 and 9 of reprogramming, 5,000 to 10,000 transduced eGFP⁺ cells expressing CD45 and/or H LA-DR, were FACS-purified and resuspended in PBS containing 0.04% bovine serum albumin (BSA). Day 0 controls were purified from eGFP-transduced cultures based on eGFP positivity at day 9 and processed similarly. Cells were loaded on a 10x Chromium (10x Genomics) without multiplexing. Single cell RNA libraries were prepared using the Chromium Single Cell 3⁺ v2 Reagent Kit (10x Genomics). Indexed sequencing libraries were quantified with a High Sensitivity DNA analysis kit (Agilent) and Agilent Bioanalyzer. Indexed libraries were pooled in an equimolar ratio and sequenced with an Illumina NextSeq 500 platform.

Analysis of single cell RNA sequencing of human cancer cells reprogrammed in 2D and 3D

10x Genomics cellranger Single Cell Software v7.1.0 was used for demultiplexing, alignment (hg38), UMI counting and single cell 3' end gene counting. The sparse expression matrix generated by cellranger analysis pipeline was used as input to Seurat library v4.3.0, and cells and genes passing quality control thresholds were included according to the following criteria: 1) total number of unique molecular identifiers detected per sample greater than 3 lower median absolute deviations (MADs); 2) number of genes detected in each single cell greater than 3 MADs; 3) percentage of counts in mitochondrial genes less than 10%. Data was normalized using "LogNormalize" with the scale factor of 10,000 and 7000 variable features were identified. For differential expression analysis between day 0 and corresponding days, FindMarkers function with Wilcox Rank Sum test was used with the following parameters: logfc.threshold = 0.1, min. pct = 0.25. Gene ontology (GO) and Reactome pathway enrichment analyses were performed using enrichR library v3.1 (https://cran.rproject.org/web/packages/enrichR/index.html). To estimate endogenous expression of SPI1, IRF8 and BATF3, the number of reads in the 5’ untranslated region (UTR) and 3’ UTR of corresponding transcription factors was calculated using multicov from bedtools v2.27.0 (https://bedtools.readthedocs.io). For total expression, multicov software was also used considering the window [start of the gene - end of the gene]. The percentage of overlapping genes between a tumor-APC signature gene list established previously (Zimmermannova et al. 2023) and differentially expressed genes in 2D and 3D settings was calculated. scPred with default svm radial model and publicly available DC single cell expression data were used for DC subset affiliation (Alquicira-Hernandez et al. 2019, Ferreira et al. 2023). Training of classifiers with available DC data was performed using 7,000 variable genes. Normalized expression levels were then classified from the reprogramming dataset. A probability threshold of 0.95 to was used classify cells into classes. GSEA was performed using singleseqgset R library (https://arc85.github.io/singleseqgset/) with default parameters. The immunogenic and tolerogenic gene signatures were based on combination of genes in Cluster 49 and Cluster 61 (TLR-induced maturation) and Cluster 103 (Homeostatic maturation) previously defined (Ardouin et al. 2016). The resulting enrichment scores were scaled in each sample individually.

Human cancer cell line-derived spheroid co-cultures with PBMC Donor-derived human PBMCs (STEMCELL Technologies) were resuspended in RPMI complete medium at the concentration of 1x105 cells/ml. When stated, medium was supplemented with IL-2 (50 U/ml; Peprotech) and IL-7 (10 ng/ml; Peprotech) to maintain T cells in culture, or with a combination of anti-CD3 (125 ng/ml; Biolegend) and anti-CD28 (250 ng/ml; Biolegend) to induce T cell pre-activation. Subsequently, 5x10³ non-activated or pre-activated PBMCs were added to the each well containing eGFP+ T98G spheroids aggregated with reprogrammed CD45+ or mCherry- transduced T98G-eGFP+ cells mixed with CAFs. PBMCs and the T98G cancer cell line were H LA-matched. Changes in size of spheroids were evaluated after 9 days of coculture with PBMCs by assessing eGFP fluorescence using Spark Multimode Microplate Reader (TECAN Life Sciences). Using PBMCs in co-culture with spheroids containing reprogrammed or eGFP-transduced T98G-eGFP+ cells mixed with CAFs, the supernatant was collected and processed with LEGENDplexTM Human CD8/NK Panel kit (Biolegend) according to the manufacturer's protocol. Cytokine concentrations were determined using the LEGENDPlex Data Analysis software (Biolegend).

Results

The inventors first generated spheroids from T98G and A375 human cancer cell lines and investigated cDC1 reprogramming efficiency 9 days after transduction. It was observed that cDC1 reprogramming progressed in the context of 3D spheroid and that reprogramming efficiency was higher in spheroids than in monolayer for the T98G cell line (Fig. 6A), indicating that the 3D environment does not compromise but rather can favour reprogramming. To verify the establishment of a transcriptional cDC1 program and uncover potential differences between 2D and 3D, the inventors performed scRNA-seq of reprogrammed T98G cells at days 3, 7, and 9. When compared to 2D cultures, reprogrammed cells in spheroids showed higher levels of transcriptional affiliation to cDC1s and rapid activation of the tumor-APC gene signature by day 3, which was established using commonly upregulated cDC1 genes during reprogramming of 18 human cancer cell lines (Zimmermannova et al. 2023) (Fig. 6B, C). Moreover, gene set enrichment analysis for immunogenic (TLR-induced maturation) or tolerogenic (homeostatic maturation) signatures (Ardouin et al. 2016) demonstrated that during reprogramming in 3D, PIB induced an immunogenic program (Fig. 6D, E). To evaluate the impact of immunosuppressive human TME components in cDC1 reprogramming, the inventors initiated reprogramming in spheroids containing increasing proportions of cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSC), or pericytes (Fig. 6F). The presence of CAFs, MDSCs, or pericytes, anti-inflammatory cytokines IL-6, TGF-p and VEGF, while the addition of antiinflammatory cytokines IL-6, TGF-p, VEGF and immuno-regulatory GM-CSF only marginally reduced reprogramming (Fig. 6F, G).

Conclusion

These results demonstrate that cDC1 reprogramming in spheroids accelerates the kinetics of reprogramming eliciting an immunogenic cDC1 cell state and indicate that the progression of cDC1 reprogramming and function in spheroids lead to efficient immune cell activation and cytotoxicity within the human T E independently of immunosuppression.

Example 8. Adenoviral vectors allow efficient delivery of cDC1 reprogramming factors to tumors.

Background

The inventors aimed to identify a platform to deliver PIB factors to tumors and elicit in situ cDC1 reprogramming. The lentiviral vector (LV-PIB-eGFP) was compared with non-integrative and replication-deficient adenoviral vector serotype 5 (Ad-PIB-eGFP) and adeno-associated viral vectors (AAV-PIB-eGFP) in monolayers, spheroids, and tumors in situ. Methods

Patient-derived cancer cell co-cultures with HLA-matched T cells

Patient-derived melanoma cells transduced with adenoviral vector serotype 5 Ad-Pl B- eGFP or adenoviral vector serotype 5 Ad-eGFP in monolayer or spheroids at reprogramming day 9 were used for co-culture experiments. For 2D cultures, transduced cells were collected in 5 ml tubes, resuspended to a concentration of 0.5x10⁶ cells per ml of RPMI complete medium supplemented with 1 mM of MART 1 ELAGIGILTV (JPT, SP-MHCI-0006) and CMV peptide NLVPMVATV (JPT SP-MHCI- 0005) and incubated for 3 hours at 37°C and 5% CO2. Purified HLA-A2-matched CD8+ T cells were added to a 48-well plate and peptide-loaded cells were washed in complete RPMI and gently loaded on top of CD8+ T cells to a final ratio of 1 transduced melanoma cell to 10 CD8+ T cells in a final volume of 500 pl. For spheroids, medium was replaced by fresh RPMI media containing 1 mM of MARTI ELAGIGILTV and CMV NLVPMVATV peptides, and spheroids were incubated for 3 hours at 37°C and 5% CO2. Peptide-loaded spheroids were then gently washed in complete RPMI and 0.5x10⁶ CD8+ T cells loaded on top using a final volume of 200 pl. After 3 and 5 days of co-culture, culture medium was replaced by fresh complete RPMI medium containing 50 U/ml of IL-2 and 10 pg/ml of IL-7. After 8 days of co-culture, cells from 2D and 3D cultures were processed for flow cytometry analysis.

Results

First, the inventors observed that reprogramming efficiency and MHC-I expression was higher with Ad and LV when compared to AAV vectors (Fig. 7A-C). The inventors then profiled reprogramming kinetics and detected rapid phenotypic changes at day 3 with LV and Ad vectors (Fig. 7D). In contrast, AAV showed low reprogramming efficiency at early time points, which did not reach the efficiencies of LV or Ad vectors. In patient- derived cancer cells, Ads were comparable to LVs in inducing cDC1 reprogramming and expression of HLA-ABC and CD40 in 2D and 3D (Fig. 7E, F). To evaluate tumor transduction capacity in situ, two consecutive intratumoral injections of LV-eGFP, adenoviral vector serotype 5 Ad-eGFP, and AAV-eGFP vectors were performed into B16 tumors and the percentages of eGFP⁺ tumor cells were quantified (Fig. 7G). The inventors observed high in situ transduction capacity with adenoviral vector serotype 5 Ad-eGFP and AAV-eGFP when 10⁹ (2.94±2.9%) and 10¹° (2.44±5.8%) viral particles or 8x10¹⁰ genomic copies (GC) were administered, respectively (Fig. 7H). In contrast, the transduction capacity of LV-eGFP was low even at the highest dose (0.3±0.2%). Adenoviral vector serotype 5 Ad-Pl B-eGFP or a control adenoviral vector serotype 5 Ad vector (Ad-Stuffer-eGFP) were then injected intratumorally into two human xenograft models (A2058 melanoma and SKLMS1 sarcoma) and transduction (eGFP⁺ 5.0±2.7% SKLMS1 ; 0.3±0.2% A2058) and in situ reprogramming (CD45⁺HLA-DR⁺ 3.9±0.7% SKLMS1; 16.6±2.5% A2058) were detected (Fig. 7I).

To complement the validation of Ad-mediated PIB delivery, the inventors evaluated antigen presentation function in patient-derived cancer spheroids. Reprogrammed patient-derived melanoma cells induced activation and expansion of H LA-matched CD8⁺ T cells, giving rise to effector CCR7'CD45RA' and cytotoxic CD95⁺CD8⁺ T cells (Fig. 7J), which also resulted in higher T cell infiltration into spheroids (Fig. 7K).

Conclusion

These data demonstrate that Ad vectors combine fast and efficient reprogramming with in situ tumor transduction capacity, providing a delivery platform for a cancer gene therapy approach based on in situ cDC1 reprogramming.

Example 9. Treatment with adenoviral vector encoding cDC1 reprogramming factors induces systemic and durable antitumor immunity

Background

The inventors aimed at profiling in vivo efficacy of a gene therapy based on in situ cDC1 reprogramming mediated by Ad vectors.

Results

The inventors established B16 tumors and administered 4 intratumoral injections of either adenoviral vector serotype 5 Ad-Pl B, adenoviral vector serotype 5 Ad-Stuffer or PBS at days 7, 9, 11 and 13 combined with anti-PD-1 and anti-CTLA-4 (Fig. 8A). Strikingly, 50% CRs were observed in mice treated with adenoviral vector serotype 5 Ad-Pl B, which remained tumor-free for 100 days (Fig. 8B). Within tumors, the inventors detected an enrichment of CD45+ cells, primarily CD8+ T cells (Fig. 8C), which negatively correlated with the tumor volume (Fig. 8D). Adenoviral vector serotype 5 Ad- PIB-treated tumors were enriched for effector T-bet+PD-1-CD8+ T cells and showed reduction in terminally exhausted T-bet-PD-1+CD8+ T cells (Fig. 8E). The ratio of pro- inflammatory CD4+ T helper cells to regulatory T cells was also elevated in Ad-Pl B- treated mice, confirming the shift towards a pro-inflammatory response in the gene therapy setting (Fig. 8F). Moreover, in both tdLN and non-draining lymph nodes (ndLN), higher frequencies of p15E-specific CD8+ T cells was detected (Fig. 8G). To test whether treatment with the adenoviral vector serotype 5 Ad-PIB gene therapy induces immunological memory, survivor mice were re-challenged subcutaneously with parental B16 cells (Fig. 8A). While naive mice developed tumors within 20 days, survivors remained tumor-free for another 60 days (Fig. 8H). In the peripheral blood of survivors, cytotoxic effector memory IFN-y+CD62L-CD44+CD8+ T cells specific for the B16 melanoma antigen PMEL were also detected (Fig. 8I). Finally, the inventors tested whether the gene therapy-induced systemic immune memory confers protection against metastatic tumor growth. Thus, it was taken advantage of the metastatic properties of B16 cells to colonize the lung and injected them intravenously at day 160 (Fig. 8A). Remarkably, survivor mice previously re-challenged subcutaneously also did not develop metastatic foci in the lungs (Fig. 8J).

Finally, the inventors investigated abscopal effect after intra-tumoral administration of PIB-encoding adenoviral vectors. B16 tumors were established on both flanks of mice and injected Ad5-PIB or Ad5-Stuffer in one tumor per mouse. Then tumor growth of treated and non-treated tumors was followed overtime. The inventors observed that 5 out of 11 tumors treated with Ad5-PIB responded to treatment and showed a strong control of tumor growth (Fig. 8K). Interestingly, control of tumor growth was also observed in contralateral non-treated tumors in responder mice, associated with significant extended survival of PIB-treated groups with 2 mice showing complete responses of both contralateral tumors (Fig. 8L, M).

Conclusion

These results support an cDC1 reprogramming gene therapy modality based on in situ delivery of Ad vectors encoding PU.1, IRF8 and BATF3 for cancer immunotherapy which triggers systemic tumor-antigen specific T cell responses that lead to long-term antitumor immunity and efficacy in non-treated tumors.

Example 10. Maintaining high in vivo transduction with gene therapy product overtime allows extended in vivo efficacy

Background The inventors aimed at assessing transduction efficiency and in vivo efficacy in MHC^LOW T-Cell^LOW B16 tumors treated with 1, 2 or 4 intratumoral injections of Ad gene therapy vector.

Results

To investigate the impact of different numbers of intratumoral injections on in vivo transduction efficiency, B16 tumors were treated with 1 , 2 or 4 intratumoral injections of eGFP-encoding adenoviral vectors and transduction was analyzed 9 days after the first injection. It was observed that administration of 4 intratumoral injections yields higher percentage of GFP+ transduced cells when compared to 1 or 2 injections (Fig. 9A). Then, the inventors investigated tumor growth and survival after 1, 2 and 4 intratumoral injections of Ad5-PIB, the control vector Ad5-Stuffer or PBS, in combination with anti-PD-1 and anti-CTLA-4 (Fig. 9B). First, it was observed that mice treated with 1 , 2 and 4 injections of Ad5-PIB showed lower tumor volume in comparison to their respective control (Analysis of area under the tumor growth curves: 1 injection, Ad5-PIB (4270 ± 1308) vs Ad5-Stuffer (9890 ± 2127); 4 injections, Ad5-PIB (2020 ± 670) vs Ad5-Stuffer (5892 ±1114)), suggesting that 1 intratumoral injection of Ad5-PIB is sufficient to induce in vivo efficacy (Fig. 9C, D). However, the inventors observed that 4 injections allowed the highest in vivo efficacy and complete responders with long term tumor-free survival (Fig. 9E).

Conclusion

In situ cDC1 reprogramming mediated by 1, 2, and 4 injections induced in vivo efficacy. However, higher number of intra-tumoral administrations of gene therapy vector maximize in vivo transduction overtime and allows higher in vivo efficacy associated with complete tumor regression.

Example 11. The constitutive Spleen Focus-Forming Virus (SFFV) promoter induces high and consistent cDC1 reprogramming efficiency across human cancer cell types.

Background

The Spleen Focus-Forming Virus (SFFV) promoter was identified as the most efficient to induce cDC1 reprogramming after transduction of Clec9a-tdTomato (tdT) reporter mouse embryonic fibroblasts with lentiviral particles encoding PIB driven by Dox- inducible (TetO) or constitutive (UbC, SFFV, PGK, EF1S, EF1 and EF1 i) promoters (Rosa et al. 2022). Before locking the SFFV promoter for the final gene therapy vector, the inventors decided to test 10 alternative promoters widely used in the clinic for their ability to induce cDC1 reprogramming.

Results

The inventors first observed that SFFV allows overall high reprogramming efficiency across cell lines (Fig. 10A). Other promoters including CMV, hPGK, MNDU3, RSV and CBA, also showed high reprogramming efficiency in specific cell lines. It was also observed that tumor-specific promoter TERT allowed cDC1 reprogramming in A2058 and SK-LMS-1 , suggesting that reprogramming can be induced by overexpression of PIB mediated by cancer-specific promoters. To further support the choice of promoter for the final gene therapy vector candidate, reprogramming efficiency mediated by constitutive promoters allowing high cDC1 reprogramming (>20% CD45 and/or HLA- DR+ cells) at low multiplicity of infection (1x10²- 1x10³1 FU/cell) was investigated using primary samples of breast, melanoma, and colorectal cancer (Fig. 10B). The inventors observed that SFFV allows high reprogramming efficiency consistently across primary cancer samples and that none of the alternative promoters (CMV, hPGK, MNDU3, RSV and CBA) slowed a consistent benefit over SFFV.

Conclusion

Considering the consistency of SFFV promoter across tested cell types and the in vitro and in vivo efficacy data generated so far with the same promoter, it was decided to lock SFFV for the final gene therapy vector candidate.

Example 12. The mutated derivative mut6 of Woodchuck Hepatitis Virus Post- Transcriptional Regulatory Element (mut6WPRE) allows higher cDC1 reprogramming efficiency.

Background

The Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is typically included in the 3'UTR (untranslated region) in the expression cassette of gene therapy vectors to enhance mRNA stability and protein yield. Because wild type WPRE contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX) previously implicated in the development of liver tumors (Kingsman et al. 2005/ the inventors investigated whether WPRE sequence downstream the FIB tricistronic cassette is required for efficient reprogramming and whether the mut6WPRE, a version of WPRE that carries a mutation disturbing the expression of the WHX protein considered safe from the regulatory perspective (Zanta- Boussif et al. 2009/ could be used instead for the gene therapy product.

Results

The inventors observed that adenoviral vectors containing WPRE sequence allowed higher reprogramming efficiency measured by surface expression of CD45 and HLA- DR mediated by Ad5 and Ad5/F35 at reprogramming day 3 when compared to adenoviral vectors without WPRE sequence (Fig. 11 A). Next, the impact of replacing the wild type WPRE sequence by the mut6WPRE was investigated. The inventors detected higher frequency of reprogrammed cells co-expressing CD45 and H LA-DR using mut6WPRE when compared to native WPRE in the melanoma A2058 (63.7%±1.2 vs 30.25%±5.5) and sarcoma SK-LMS-1 (23.5%±2.55 vs 3.18%±0.88) cell lines at MOI 1x10³ IFU/cell (Fig. 11B, C).

Conclusion

These data suggests that WPRE sequence downstream the PIB tricistronic cassette is required for higher cDC1 reprogramming efficiency and supports the selection of mut6WPRE sequence for the final gene therapy vector candidate.

Example 13. The Rabbit beta-globin polyadenylation signal (rBGpA) Post- Transcriptional Regulatory Element enhances cDC1 reprogramming efficiency.

Background

The polyadenylation signal downstream the WPRE sequence can affect transgene expression and thus impact cDC1 reprogramming efficiency. To identify the optimal polyadenylation signal for the final gene therapy vector, 6 polyA sequences were tested and investigated for their ability to induce cDC1 reprogramming.

Results

The inventors observed that all polyA tested were at least as efficient as the BGHpA across cancer cell lines and that rabbit Beta-Globin polyadenylation signal (rBGpA) was able to increase the percentage of reprogrammed cells at low MOI even in the more resistant to reprogram cell line Ca9-22 (Fig. 12A). It was also observed that rBGpA allows high cDC1 reprogramming in both breast and melanoma samples (Fig.

12B).

Conclusion

These data suggest rBGpA as the optimal polyA signal for efficient cDC1 reprogramming and supports the selection of rBGpA for the final gene therapy candidate.

Example 14. PIB tricistronic cassette encoding PU.1 followed by IRF8 and BATF3 allows high reprogramming efficiency.

The inventors have previously demonstrated that the order of PU.1, IRF8 and BATF3 in the tricistronic cassette can affect cDC1 reprogramming efficiency (Rosa et al. 2018). Thus, it was decided to test different versions of the PIB tricistronic cassette with all possible order of transcription factors.

Results

It was observed that all versions of the tricistronic cassette were able to induce upregulation of CD45 and HLA-DR in human cancer cell lines and primary cancer samples (Fig. 13A, B). Moreover, no consistent differences between cDC1 reprogramming capacity of PIB cassette and alternative versions were observed.

Conclusion

Together, these data suggests that order of PU.1 , IRF8 and BATF3 does not impact drastically cDC1 reprogramming efficiency. Considering that the inventors have used the PIB cassette in all previous work, these data support the selection of PIB cassette for the final gene therapy candidate.

Example 15. Optimized PIB-encoding expression cassette allows high reprogramming efficiency.

Background The inventors have previously tested different promoters, tricistronic cassettes encoding the reprogramming factors PU.1, IRF8 and BATF3 in different orders, WPRE and pA sequences, and have selected the regulatory elements 1) Spleen focus forming viral (SFFV) promoter, 2) tricistronic cassette PU.1-P2A-IRF8-T2A-BATF3, 3) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), and 3) rabbit beta-globin polyadenylation signal (rbBGpA) for the final gene therapy vector. As these elements were tested one-by-one, the inventors decided to generate adenoviral vectors encoding the full optimized expression cassette and investigate its reprogramming capacity when compared to the expression cassette previously used for in vitro efficacy studies.

Results

The inventors have observed that, not only the single elements tested, but also the full optimized expression cassette (WPREmut6-rbBGpA or AT-108) allows high reprogramming efficiency across human cancer cell lines and primary samples in monolayer and in organoids compared to the previous version (WPRE-BGHpA or Ad5- PIB) (Fig. 14 A, B, C, D). Interestingly, the optimized expression cassette containing WPREmut6-rbBGpA allowed a major increase in reprogramming efficiency in human cancer cell lines and in the primary colorectal cancer samples CRC50 at low MOI (100 I FU/cell) when compared to the expression cassette containing WPRE-BGHpA. The inventors observed that the optimized expression cassette allowed superior in vivo reprogramming yield when delivered via intra-tumoral injection (Fig. 14 E), and that reprogrammed primary cancer cells generated with the optimized cassette acquired higher antigen cross-presentation capacity in the absence of TLR stimulation (Fig. 14 F), acquired cytokine secretion capacity (Fig. 14 G) and ultimately enabled superior in vivo efficacy and animal survival in B16 model compared to the previous version (Fig. 14 H).

Conclusion

These data shows that the optimized expression cassette SFFV-PIB-WPREmut6- rbBGpA allows higher reprogramming efficiency, higher functionality of reprogrammed cancer cells and superior in vivo efficacy when compared to previous versions of the expression cassette and supports its selection for the final gene therapy vector candidate. The comparison of the reprogramming efficiency data obtained from the single elements (WPREmut6 and rbBGpA, Fig. 11 and 12) compared to said elements combined in Fig. 14 further suggests that the combination of elements provides an effect on reprogramming efficiency which is superior to the mere sum of the effects of the individual elements.

Example 16. Transduction enhancers to adenovirus serotype 5 can enhance transduction capacity in cancer cells

Background

Considering that coxsackie and adenovirus receptor (CAR), the main entry receptor of Ad5, is downregulated in subsets of solid tumors (Hensen et al. 2020), the inventors decided to test whether adding transduction enhancers to the Ad5 could enhance transduction efficiency in cancer cells. For this, 2 main variants of Ad5 were tested: 1) Ad5 with an Arg-Gly-Asp (RGD) motif introduced in the surface-exposed loop of the adenovirus fiber knob (Ad5-RGD) to enable cell entry via RGD binding integrins (Wu et al. 2002); and 2) Ad5 containing a chimeric fiber protein from Ad35 enabling cell entry via CD46 (Ad5/F35) (Mizuguchi et al. 2002)

Results

To investigate which adenoviral serotype allows higher transduction efficiency of human cells, 4 human cancer cell lines (T98G, SK-LMS-1, A2058 and Ca9-22) and 13 primary cancer cells (of colorectal, head and neck, melanoma, lung and breast tumors) were transduced with GFP-encoding Ad5, Ad5-RGD and Ad5/F35 vectors at 2 different MOIs (100 I FU/cell and 1000 I FU/cell) and transduction efficiency profiled after 3 days. Interestingly, the inventors observed that Ad5/F35 allows higher transduction efficiency across cell lines and primary samples using 100 I FU/cell (Fig. 15). We also noted that Ad5-RGD showed a tendency to allow higher transduction efficiency in primary samples, particularly at 100 I FU/cell.

Conclusion

Together, these data suggests that addition of transduction enhancers to Ad5 serotype, such as Ad5/F35 and Ad5-RGD modifications, enhance transduction efficiency on human cancer cells. Example 17. Transduction enhancers to adenovirus serotype 5 enhance cDC1 reprogramming efficiency in cancer cells

Background

Given the increase in transduction efficiency observed in cancer cells when Ad5-RGD and Ad5/F35 vectors were used when compared to Ad5, the inventors decided to compare cDC1 reprogramming efficiency induced by PIB-encoding Ad5, Ad5-RGD, Ad5/F35 and Ad5 vector containing a chimeric fiber protein from Ad3 enabling cell entry via CD46 and/or Desmoglein 2 (DSG-2) (Ad5/3) (Fig. 16C). The skilled person will appreciate that several factors can influence the intracellular expression, and functionality of the reprogramming factors intracellularly, and thus that increased transduction efficiency may not necessarily correlate with increased reprogramming.

Results

4 human cancer cell lines (T98G, SK-LMS-1 , A2058 and Ca9-22) and 3 primary cancer cells (of melanoma, head and neck and colorectal tumors) were transduced with PIB- encoding Ad5, Ad5-RGD, Ad5/F35 and Ad5/3 vectors (SEQ ID Nos: 1-4) at 2 different MOIs (100 I FU/cell and 1000 I FU/cell) and cDC1 reprogramming efficiency after 3 (cell lines) or 9 days (primary samples) was profiled. Interestingly, the inventors observed that Ad5/F35 and Ad5/3 allow >100-fold increase in reprogramming efficiency in cell lines at a MOI of 10 I FU/cell, demonstrating their ability to induce efficient cDC1 reprogramming at low MOIs (Fig. 16A). The same trend was observed in primary samples, with Ad5 vector with transduction enhancers allowing overall higher reprogramming efficiency (Fig. 16B). Ad5/F35 vector stand out as the serotype allowing consistently higher reprogramming efficiency across human cancer samples.

Conclusion

These data demonstrate that addition of transduction enhancers to Ad5 serotype also facilitate higher cDC1 reprogramming efficiency in human cancer cells.

Example 18. Lower tumor mutational burden and microsatellite stability status associate with higher reprogramming efficiency

Background The inventors have previously demonstrated that primary tumor cells can be reprogrammed into antigen-presenting cDC1-like cells through viral delivery of the transcription factors PU.1 , IRF8, and BATF3. However, in vivo cDC1 reprogramming may be influenced by tumor specific factors, including its tissue of origin and genomic landscape. Here, the inventors investigated whether the tissue of origin or genomic features, such as tumor mutational burden (TMB) and microsatellite instability (MSI), impact cDC1 reprogramming efficiency.

Methods

Reprogramming efficiency

Reprogramming efficiency for primary samples was profiled by flow cytometry quantification of CD45 and/or H LA-DR at day 9 after transduction with lentiviral or adenoviral vectors encoding PU.1, IRF8 and BATF3.

Tumour Mutation Burden and microsatellite stability

Tumour Mutation Burden (TMB) was estimated from whole-exome sequencing (WES) data. Sequencing data were analysed using the Sarek pipeline (v3.4.4) (Hanssen et al. 2024, Garcia et al. 2020) . To distinguish germline from somatic mutations, Mutect2 (Cibulskis et al. 2013) was used in “tumour only” mode. Mutation calls were filtered to remove any mutation overlapping a known polymorphism in GnomAD v3.2.1 (Chen et al. 2024) or with a read depth lower than 10 reads. The final TMB was estimated using Maftools v2.10.05 (Mayakonda et al. 2018). To obtain estimates comparable to other samples, Mutect2 mutations after VEP (McLaren et al. 2016) annotations were filtered using the SureSelect Exon v5 bed file.

To infer microsatellite stability status of primary samples, mutations were used to infer the proportion of SBS6, SBS14, SBS15, SBS20, SBS21 , SBS26 and SBS44 MSI- associated mutation signatures (COSMIC: Catalogue of Somatic Mutations in Cancer; https://www.cosmickb.org) in each sample. The following thresholds were used to call the microsatellite stability status of the samples: MSS: % contribution of MSI- associated signatures < 20; MSI: % contribution of MSI-associated signatures > 25. Intermediate percentages of contributions translating onto an unknown MSI status.

Results

To investigate whether tumor-intrinsic genomic features influence the reprogramming efficiency of cancer cells, the inventors analyzed the relationship between tumor mutational burden (TMB), microsatellite instability (MSI) status, and the percentage of reprogrammed primary cancer cells of multiple indications including colorectal, brain, melanoma, head and neck, lung and bladder cancer. A significant inverse correlation between TMB and reprogramming efficiency was observed (Fig. 17, left panel). Additionally, MSS tumors exhibited significantly higher reprogramming efficiency compared to MSI-high tumors (Fig. 17, right panel), indicating that MSI status is a negative predictor of reprogramming efficiency.

Conclusion

Together, these findings highlight high TMB and MSI status as biomarkers associated with reduced reprogramming efficiency in primary cancer cells.

Example 19. In vivo cDC1 reprogramming elicits tumor-origin agnostic immunity across subcutaneous and orthotopic models

Background

The response rates of immune checkpoint blockade (ICB) in solid tumors, such as lung, colon, bladder, breast cancer, and glioblastoma, remains below 25%. Therefore, the inventors decided to investigate whether in vivo cDC1 reprogramming would elicit potent anti-tumor immune response across different syngeneic tumor models as a monotherapy or in combination with anti-PD-1.

Methods

In vivo efficacy experiments

To evaluate the tumor growth and survival after in vivo cDC1 reprogramming of syngeneic mouse tumor models, we transduced cancer cells in vitro with lentivirus SFFV-PIB-eGFP (expressing PU.1, IRF8, BATF3 and eGFP as a reporter of transduction) or empty lentiviral vector as a transduction control SFFV-eGFP (empty lentiviral backbone expressing eGFP) and 16 hours after transduction injected the cells into C57BL/6J or BALB/c mice as a mix of 1:1 transduced eGFP+ to untransduced eGFP- cells. The transduction was performed in the presence of polybrene (8 pg/ml). In a subset of experiments, cancer cells were transduced with adenoviral vectors encoding PIB (Ad5-PIB) or control adenoviral vectors (Ad5-Stuffer). For adenoviral vectors, cell mixtures were kept in vitro to estimate the percentages of reprogrammed cells by CD45 and MHC-II quantification at day 9, which is indicated in the figure. For subcutaneous models, injections were performed into the right flank of animals with 1 x10⁵ cells. For orthotopic models, 1x10⁵ Lewis Lung Carcinoma (LLC) was injected intravenously, 1x10⁴4T1 cells were injected into the left mammary fat pad and 1600 SB28 cells were injected intracranially into the right hemisphere. Mice received 200 pg isotype (lgG2a) or anti-PD-1 administration intraperitoneally on days 7, 10 and 13 after tumor establishment. Mice were 6- to 12-week-old age-matched females and tumor volumes were monitored with a digital caliper and calculated using the formula V = L*W*H/2. Survival was determined by predefined end points such as tumor size reaching 1500 mm³, tumor ulceration, or signs of animal suffering. Animals were randomized for tumor establishment. Mice challenged with LLC were sacrificed on day 28 and the lungs were snap-frozen in OCT for cryo-preservation and 10 pm cuts were stained by hematoxylin and eosin (H&E) and imaged on a clinical grade light microscope (Olympus Model BX43F). The tumor area was quantified as percentage per lung area using Fiji software (Version 2.9.0).

Results

In vivo cDC1 reprogramming mediated by lentiviral vectors elicited complete responses (CR) at higher rates than aPD-1 treatment, including in the colon (CT26, 100% vs 20% CR), breast (4T1 , 100% vs. 20% CR) and lung (LLC, 3.1% vs. 21.3% tumor area/total lung area) cancer models (Fig. 18A). Next, the inventors investigated in vivo cDC1 reprogramming as a monotherapy in glioblastoma SB28, which is resistant to ICB. The inventors observed increased medium survival (MS, 82.5 versus 25.5 days; P < 0.01) in this aggressive model. Moreover, the inventors also observed that in vivo reprogramming mediated by adenoviral vectors induced efficacy in monotherapy in the colon cancer MC38 model and efficacy in combination with anti-PD-1 treatment in the bladder cancer MB49 model (Fig. 18B).

Conclusion

These data suggest that in vivo cDC1 reprogramming is a tumor-origin agnostic immunotherapy platform, which as a monotherapy and in combination with ICB is able to induce long-term anti-tumor immunity across tumor models and immunity in the native environment of the tumor.

Example 20. In vivo cDC1 reprogramming recruits TIL-B cell populations to the tumor and elicits the generation of tumor-specific antibody production Background

Positive anti-tumor responses in patients treated with immunotherapy have been linked to the presence of tertiary lymphoid structures (TLS) within tumors, where sustained T and B cell priming against tumor antigens generates tumor-specific antibody responses. Therefore, the inventors decided to investigate whether in vivo cDC1 reprogramming recruits B cells to tumors and elicits maturation of B cells for the generation of systemic tumor-specific antibody responses.

Methods

Characterization of B cell populations and tumor-specific antibody binding assay To characterize B cell populations within melanoma tumors and the generation of tumor-specific antibody responses after in vivo cDC1 reprogramming, we implanted subcutaneously YUMM 1.7 cancer cells 16 h after transduction with lentivirus SFFV- PIB-eGFP or control lentiviral vector SFFV-eGFP into C57BL/6J as a mix of 1 :1 transduced eGFP+ to untransduced eGFP- cells. Established tumors were isolated at day 21 and chopped into pieces of 2 mm diameter for dissociation. To generate single cell suspensions, tumor tissue was further mechanically and enzymatically processed for 30-60 minutes at 37°C using a digestion solution of 1 mg/ml Collagenase D (Sigma- Aldrich) and 10 pg/ml DNase I (Sigma-Aldrich) in RPMI complete medium while shaking. The resulting cell suspension was passed through a 70 pm filter and divided for flow cytometry analysis using antibody staining panels for B cells (CD45+ CD19+ B220+), lgM+ B cells (CD19+ B220+ lgM+), lgG1+ B cells (CD19+ B220+ lgG1 +), activated B cells (CD19+ B220+ CD95+ GL7+), mature B cells (CD45+ CD19+ CD23+), plasma cells (CD45+ CD19- CD138+), lgG1+ plasma cells (CD19- CD138+) and follicular dendritic cells (CD45+ CD19- CD23+). For tumor-specific antibody binding assays, we isolated serum from treated animals and control animals at days 5, 9, 14, 21 , 28, and 70. We incubated the serum diluted 1 :50 in PBS with YUMM1.7 melanoma cells at room temperature. After 30 minutes of incubation, serum was washed away and fluorescently labelled antibodies to mouse IgM , I gG 1 or IgA were used for secondary staining before flow cytometry analysis for single-cell suspensions of YUMM1.7 cells or for confocal analysis of YUMM1.7 tumor tissue, respectively. Finally, the mean fluorescence intensity was quantified.

Results

Tumor immunophenotyping revealed that in vivo cDC1 reprogramming of YUMM 1.7 melanoma tumors increased the tumor infiltrating lymphocyte (TIL) B cell populations, including lgM⁺, activated B cells, plasma cells, and follicular DCs on day 21 (Fig. 19A). This resulted in the production of circulating tumor-specific IgM antibodies that emerge by day 14, followed by IgG 1 and IgA on day 21 (Fig. 19B).

Conclusion

In vivo cDC1 reprogramming generates tumor-specific antibody responses by recruiting tumor infiltrating B cell populations to the tumor. These findings may in turn be used in clinical translation of blood-based antibody biomarkers in methods for monitoring treatment efficacy and/or patient response.

Example 21. Treatment regimen with a 3-injection lead cycle followed by 2 boost injections enable in vivo efficacy in B16 model.

Background

AT-108 treatment after 4 intratumoral injections have demonstrated robust in vivo efficacy in a MHC^LOW, Tcell^LOW mouse syngeneic model in combination with immune checkpoint blockade (Ascic et al 2024). However, to gain insight of in vivo efficacy following a dose schedule reflecting planned Fl H-trials, the inventors conduced a doseresponse in vivo efficacy experiment and performed a lead cycle of 3 injections at days 0, 2 and 4, followed by boost injections at days 9 and 14. An additional objective was to investigate AT-108 as a monotherapy treatment.

Results

The inventors treated subcutaneous B16 tumors with PBS or AT-108 via intratumoral injection at day 0, 2, 4, 9 and 14 in combination with immune checkpoint inhibitors including anti-PD1 and anti-CTLA-4 (ICB) at day 0, 3 and 7. The different doses of AT- 108 tested included 10⁸, 10⁹ and 10¹° viral particles (VPs) per dose (Fig. 20A). The inventors observed that the lowest dose of 10⁸ VPs was sufficient to induce tumor growth delay. Moreover, the inventors observed that 10⁹ VPs was the minimal dose required to induce complete tumor regression (Fig. 20B). Quantification of tumor volumes at day 14 showed significant control of tumor growth at all doses compared to control PBS group (Fig. 20C). However, we noted that the highest dose (10¹° VPs) allowed the highest percentage of mice with complete tumor regression. All doses significantly extended survival in comparison to control group (Fig. 20D).

Next, the inventors investigated in vivo efficacy after monotherapy treatment with AT- 108 following the same dosing schedule described above (Fig. 21 A). Monotherapy treatment with AT-108 allowed control of tumor growth, already observed at day 10 after first dose (Fig. 21 B, C), and enabled significant prolonged survival compared to both PBS- and PBS+ICB- treated groups (Fig. 21 D). Importantly, we also observed a dose-response in monotherapy, with the highest dose (10¹° VPs) allowing highest extended survival (Fig. 21 E).

Conclusion

Together, these data demonstrate a clear dose-response relationship between AT-108 and in vivo efficacy using a treatment regimen composed of a lead cycle with 3 injections followed by 2 boost injections. Importantly, the inventors observed that AT- 108 induced in vivo efficacy as monotherapy in B16 model.

Example 22. One intra-tumoral injection of Ad5-PIB is sufficient to induce in vivo efficacy, further improved by multiple administration

Background

AT-108 treatment after 4 intra-tumoral injections enables in vivo efficacy. To investigate if a lower number of injections is sufficient to induce complete tumor regression, the inventors performed an in vivo efficacy experiment to compare efficacy mediated by 1, 2, 3 or 4 injections of Ad5-PIB (Fig. 22A).

Results

As observed in Example 10, 1 injection of Ad5-PIB was sufficient to induce in vivo efficacy (Fig. 22B). However, in this example, in vivo efficacy was further improved with 3 injections and this was the minimal requirement for complete tumor clearance. 3 injections induced comparable in vivo efficacy to 4 injections.

Conclusion

Together, these data demonstrate a dose-response relationship between Ad5-PIB and dosing intensity.

Example 23. Weekly boost injections of AT-108 significantly improve mice survival.

Background AT-108 treatment after a lead cycle of 4 intra-tumoral injections was shown to induce robust in vivo efficacy in the B16 melanoma mouse syngeneic model in combination with immune checkpoint blockade. Here, the inventors wanted to investigate whether weekly boost injections after initial lead treatment cycle enables higher in vivo efficacy.

Results

To investigate survival benefits of repeated weekly boost injections of Ad5-PIB after the lead treatment cycle, the inventors performed 3 intra-tumoral injections of Ad5-PIB at day 0, 2 and 7 (lead cycle), followed by weekly boost injections at days 14 (week 2), 21 (week 3), 28 (week 4) and 35 (week 5) in the B16 model. Mice were also treated with immune checkpoint blockade (anti-PD1 and anti-CTLA-4, ICB) at day 0, 3 and 7 by intraperitoneal injection (Fig. 23A). The inventors observed that boost cycles with Ad5- PIB allowed a significant improvement in overall and median survival (median survival: Ad5-PIB, 25 days; Ad5-PIB+boost, 52 days) (Fig. 23B). The inventors also observed that the first boost-cycle allowed tumor regression in one animal.

Conclusion

Together, these data show that performing weekly boost injections of Ad5-PIB after the lead treatment cycle allow sustained in vivo efficacy and extended survival.

Additionally, the data suggests that AT-108 delivery is not effected by pre-existing Ad5 immunity.

Example 24. AT-108 induces abscopal effect in B16 tumor model

Background

Intra-tumoral delivery of adenoviral vectors encoding PU.1 , IRF8 and BATF3 (Ad5-PIB) to B16 murine melanoma tumors in combination with immune checkpoint blockade (ICB, anti-PD1 and anti-CTLA4 antibodies) treatment has shown significant benefit, leading to complete tumor regression (Ascic et al. 2024). Here, the inventors tested whether Ad5-PIB in combination with ICBs (anti-PD1 and anti-CTLA-4) could induce systemic immunity and efficacy in contra-lateral non-treated tumors and profiled its mode of action.

Results

To evaluate whether local treatment and in situ cDC1 reprogramming induces systemic immunity and allows efficacy in distal, non-treated tumors in combination with anti-PD1 and anti-CTLA4 antibodies (ICB, dosed at days 0, 3 and 6), the inventors have established murine B16 tumors on both flanks of mice, injected Ad5-PIB or Ad5-Stuffer in one tumor per mouse (dosed at days 0, 2, 4 and 6) and followed tumor growth of treated tumors to observe local responses and distal non-treated tumors to assess abscopal effect.

First, the inventors classified PIB-treated mice as responders (R PIB) and nonresponders (NR PIB) by determining the percentage of change in volume of treated tumors from baseline/day 0 to day 30 post first treatment. Treated tumors of responder mice were characterized by a >30 % decline in tumor volume from baseline. From 11 tumors treated with Ad5-PIB, 5 tumors responded to treatment and showed a strong control of treated tumor growth in comparison to tumors treated with the control Ad5- Stuffer vector (Fig. 24A, B). Control of tumor growth was observed in non-treated tumors, suggesting that in situ cDC1 reprogramming mediated by the gene therapy product allows abscopal effect. Moreover, PIB-treated mice showed significant extended survival until day 200 with 3 mice showing complete tumor regression of both tumors (Fig. 24C).

To investigate the mode of action underlying abscopal effect, the inventors performed immune profiling of tumors 9 days after treatment initiation in the same abscopal setting. Ad5-PIB increased the overall immune infiltrates in treated and non-treated tumors (Fig. 25). An increase in CD45+ cells (1.6x) (Fig. 25A), CD8+ T (1.8x) (Fig. 25B) and CD4+ T cells (2x) (Fig. 25C), NK cells (2x) (Fig. 25D), and CD19+ B cells (2x) (Fig. 25E) was observed in abscopal non-treated tumors isolated from PIB-treated mice, when compared to Stuffer-treated groups. Phenotypically, these CD8+ T cells presented a more activated effector phenotype (T-bet+PD-1-) and less terminally exhausted (T-bet-PD-1+) in tumors treated with PIB and in the abscopal tumors (Fig. 25B). Moreover, a decreased in regulatory CD25+CD4+ T cells was observed in both treated and non-treated tumors in Ad5-PIB treatment groups compared to Ad5-Stuffer groups, as well as an increase in Th1/effector CD4+ T cells (Fig. 25C). Interestingly, infiltration of CD8+, CD4+ and NK cells in the treated site correlated with their infiltration in the non-treated site (Fig. 25F).

Conclusion In situ cDC1 reprogramming induces control of tumor growth in injected and nontreated distal tumors, allowing complete regression of both tumors and long-term survival. Efficacy was associated with increased infiltration of immune cells (CD45+), CD8+ and CD4+ T cells, NK cells, and B cells in treated and non-treated tumors. CD8+ T cells showed increased effector phenotype, while regulatory CD4+ T cells were reduced in tumors treated with Ad5-PIB and in the contralateral non-treated tumors, compared to Ad5-Stuffer treatment groups. In summary, Ad5-PIB treatment induces a more inflamed tumor microenvironment in both treated and non-treated tumors.

Example 25. Characterization of in vivo efficacy of AT-108 in YUMM1.7 in monotherapy and combination with anti-PD-1

Background

Here, the inventors investigated the anti-tumor efficacy of AT-108 in YUMM1.7 in monotherapy and combination with anti-PD-1 (aPD-1) treatment.

Results

YUMM 1.7 tumors were established in C57BL/6J mice and treated with 4 intra-tumoral injections of PBS or AT-108 at day 0, 2, 4 and 7 as monotherapy or in combination with aPD-1 (day 0, 3 and 7). Treatment with AT-108 in monotherapy showed control of tumor growth and extended survival (2 complete responders out of 15) compared to controls (Fig. 26A, B). Combination with aPD-1 treatment further improved control of tumor growth and mice survival, leading to 7 out of 15 complete responders. Interestingly, the inventors could detect expansion of tumor-reactive T cells as early as 10 days post first dose of AT-108 (Fig. 26C).

Conclusion

AT-108 shows anti-tumor efficacy in YUMM 1.7 melanoma tumors in monotherapy, including complete responders (2/15), as well as in combination with aPD-1 (7/15 complete responders). Moreover, AT-108 induces expansion of tumor-reactive T cells detected in peripheral blood.

Example 26. AT-108 Demonstrates In Vivo Efficacy in the PANC02 Pancreatic Cancer Model

Background In this example, the inventors assessed the anti-tumor activity of AT-108 in a syngeneic pancreatic cancer model. The study was designed to evaluate the therapeutic efficacy of AT-108 in combination with immune checkpoint blockade using anti-PD-1 (aPD-1) therapy.

Methods

Subcutaneous PANC02 tumors were established in immunocompetent C57BL/6J mice. Mice received four intra-tumoral injections of either PBS or AT-108 on Days 0, 2, 4, and 7. Treatment with aPD-1 was administered on Days 0, 3, and 7. Tumor progression was monitored over time, and therapeutic efficacy was assessed by tumor volume measurements.

Results

Combination treatment with AT-108 and aPD-1 led to significant tumor growth inhibition compared to control groups, indicating that AT-108 synergizes with aPD-1 to mediate effective anti-tumor responses in the PANC02 model (Fig. 27).

Conclusion

AT-108 induces potent anti-tumor activity in a pancreatic cancer model when administered in combination with aPD-1 immune checkpoint inhibition. These results support the potential application of AT-108 in solid tumors with poor baseline immunogenicity, such as pancreatic cancer.

Example 27. Inclusion of 2 maintenance cycles enhance in vivo efficacy

Background

In this example, the inventors evaluated the impact of treatment scheduling on the in vivo efficacy of AT-108. Specifically, they tested whether extending a standard lead dosing regimen (three injections on Days 0, 2, and 4) with two additional maintenance cycles (booster cycles) could improve therapeutic outcomes. In parallel, they assessed whether a less intensive dosing schedule — four injections administered every five days — could also confer efficacy while potentially reducing treatment burden.

Methods

B16 tumors were established in immunocompetent C57BL/6J mice and treated with intra-tumoral injections of either PBS or AT-108. Mice received either (i) a lead cycle of three injections on Days 0, 2, and 4, (ii) a lead cycle of three injections on Days 0, 2, and 4 followed by two maintenance doses (booster cycles) on Days 9 and 14, or (iii) four evenly spaced injections delivered every five days. All treatment arms were administered in combination with aPD-1 and aCTLA-4 checkpoint inhibitors on Days 0, 3, and 7. Efficacy was assessed through tumor growth monitoring and survival analysis.

Results

Addition of two maintenance cycles (booster cycles) to the lead dosing regimen enhanced early anti-tumor responses and extended median overall survival compared to the lead cycle alone (Fig. 28). Lower-intensity regimen consisting of four injections spaced every five days also demonstrated efficacy, with some animals achieving complete tumor regression. However, the most robust in vivo efficacy was observed with the regimen comprising a lead cycle of 3 doses followed by two maintenance doses (booster cycles) (Fig. 29).

Conclusion

These results demonstrate that AT-108 achieves maximal in vivo efficacy when administered as a lead cycle of three injections followed by two maintenance cycles. Additionally, a less intensive dosing schedule of four injections every five days is sufficient to drive meaningful anti-tumor responses, offering flexibility for alternative therapeutic protocols.

Example 28. Intra-Peritoneal Delivery of AT-108 Enables In Vivo Efficacy in an Ovarian Cancer Ascites Model

Background

In this example, the inventors evaluated whether the therapeutic efficacy of AT-108 could be achieved in vivo when delivered into anatomically confined spaces such as the peritoneal cavity. The rationale was that local retention of the viral vector within such compartments may promote enhanced transduction and therapeutic activity, even in the absence of active targeting.

Methods

An ovarian cancer ascites model was established in immunocompetent C57BL/6J mice through intraperitoneal injection of ID8-luciferase (ID8-luc) cells. Tumor burden was assessed by bioluminescence imaging, and animals were randomized on Day 0 based on equivalent radiance signals (average ~4 * 10⁶ photons/sec/cm²/sr). Mice received three initial IP injections of either PBS or AT-108 on Days 0, 2, and 4, followed by two maintenance doses (booster cycles) on Days 9 and 14. Treatment was administered either as monotherapy or in combination with aPD-1 checkpoint blockade, delivered on Days 0, 3, and 7 (Fig. 30A). Therapeutic efficacy was determined by longitudinal radiance monitoring, with statistical analyses performed using data collected on Day 36 post-randomization.

Results

AT-108 monotherapy led to a measurable reduction in tumor burden, as indicated by decreased radiance values relative to control-treated animals. Furthermore, combination therapy with AT-108 and aPD-1 resulted in enhanced anti-tumor activity, supporting a synergistic interaction between the two agents (Fig. 30B).

Conclusion

These findings demonstrate that intraperitoneal administration of AT-108 is effective in reducing tumor burden in an ovarian cancer ascites model, both as a single agent and in combination with immune checkpoint blockade. The results support the strategy of delivering AT-108 into confined anatomical spaces, where passive retention may bypass the need for active vector targeting to achieve therapeutic efficacy.

Example 29. AT-108 allows dose-dependent functional reprogramming in patient- derived samples.

Background

In this example, the inventors investigated the relationship between viral dose and the efficiency of cellular reprogramming, as well as the acquisition of functional characteristics associated with conventional type 1 dendritic cells (cDC1s). The study was conducted using both 2D monolayers and 3D organoid cultures derived from primary human tumors, including melanoma, head and neck cancer, and colorectal cancer.

Methods

Cytokine secretion analysis Quantification of immune-stimulatory cytokines in cell culture supernatants was performed using cytometric bead array and flow cytometry analysis. In summary, primary melanoma cells transduced with AT-108 (day 8) were stimulated overnight with TLR agonists (TLR3 (Poly l:C), TLR4 (LPS) and TLR7/8 (R848)). Cytokine secretion was profiled at day 9. Six melanoma patients were used.

Antigen cross-presentation

Primary melanoma cells transduced with AT-108 (day 8) were pulsed with long MART- 1 peptide and stimulated overnight with TLR agonists (TLR3 (Poly l:C), TLR4 (LPS) and TLR7/8 (R848)). Antigen cross-presentation was evaluated 8 days after co-culture with HLA-A2+ MART-1+ CD8+ T cells as % MART-1 tetramer+ T cells (3 HLA-A2+ patients).

Allogeneic T cell activation using mixed lymphocyte reaction

Primary cancer cells transduced with AT-108 were co-cultured with allogeneic T cells at reprogramming day 9. T cell activation was evaluated by flow cytometry quantification of CD69+ and PD1+ T cells 3 days after co-culture and IFNy+ T cells 1 day after coculture.

Autologous T cell activation and cytotoxicity

Primary cancer cells isolated from 3 melanoma patients were transduced with AT-108 and co-cultured with autologous TILs at reprogramming day 8. T cell cytotoxicity was evaluated by xCellligence assay over 3 days of co-culture.

Results

Dose-dependent induction of immune and dendritic cell markers was observed across a panel of patient-derived samples. Specifically, increased expression of CD45 and HLA-DR was detected in primary cancer cells from melanoma (n = 6), head and neck cancer (n = 4), and colorectal cancer (n = 9) (Fig. 31A). Additionally, expression of cDC1-specific markers (CD141 , CLEC9A, XCR1), HLA-ABC, and co-stimulatory molecules (CD40, CD80, CD86) also increased in a dose-responsive manner (Fig. 31A). Similar findings were reproduced in organoid models derived from head and neck (n = 3) and colorectal cancer patients (n = 3) (Fig. 31 B). Functional assays revealed that increasing doses of AT-108 led to higher secretion of I L-12p70, TNFa, CXCL10, and IFN-p (Fig. 31C), as well as enhanced antigen crosspresentation (Fig. 31 D). Reprogrammed cells further demonstrated robust allogeneic T cell activation (Fig. 31 E) and promoted autologous TIL activation, with higher doses resulting in superior tumor cell cytotoxicity (e.g., CD69: 300 vs 100 IFU for head and neck and 1000 vs 100 IFU for colorectal) (Fig. 31 F).

Conclusion

These data demonstrate that AT-108 drives dose-dependent reprogramming of patient- derived cancer cells into functional cDC1-like cells capable of initiating robust immune responses.

Sequence overview

SEQ. ID NO: 1

AT-108: Ad5-PIB

SEQ. ID NO: 2

AT-110: Ad5/F35-PIB

SEQ. ID NO: 3

AT-111: Ad5-RGD-PIB

SEQ. ID NO: 4

AT-112: Ad5/3-PIB

SEQ. ID NO: 5

SFFV-PI B-WPREmut6-rbBGpA cassette

CTGCAGCCCCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATG AAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTGCAGTAACGCCATTTTGCAAGG CATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATG AAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCG GCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCA AGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATC AGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTA ACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATA

AAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGC

CCGGGCAAGTTTGTACAAAAAAGCAGGCTGCCACCATGTTACAGGCGTGCAAAAT

GGAAGGGTTTCCCCTCGTCCCCCCTCCATCAGAAGACCTGGTGCCCTATGACAC

GGATCTATACCAACGCCAAACGCACGAGTATTACCCCTATCTCAGCAGTGATGGG

GAGAGCCATAGCGACCATTACTGGGACTTCCACCCCCACCACGTGCACAGCGAG

TTCGAGAGCTTCGCCGAGAACAACTTCACGGAGCTCCAGAGCGTGCAGCCCCCG

CAGCTGCAGCAGCTCTACCGCCACATGGAGCTGGAGCAGATGCACGTCCTCGAT

ACCCCCATGGTGCCACCCCATCCCAGTCTTGGCCACCAGGTCTCCTACCTGCCC

CGGATGTGCCTCCAGTACCCATCCCTGTCCCCAGCCCAGCCCAGCTCAGATGAG

GAGGAGGGCGAGCGGCAGAGCCCCCCACTGGAGGTGTCTGACGGCGAGGCGG

ATGGCCTGGAGCCCGGGCCTGGGCTCCTGCCTGGGGAGACAGGCAGCAAGAAG

AAGATCCGCCTGTACCAGTTCCTGTTGGACCTGCTCCGCAGCGGCGACATGAAG

GACAGCATCTGGTGGGTGGACAAGGACAAGGGCACCTTCCAGTTCTCGTCCAAG

CACAAGGAGGCGCTGGCGCACCGCTGGGGCATCCAGAAGGGCAACCGCAAGAA

GATGACCTACCAGAAGATGGCGCGCGCGCTGCGCAACTACGGCAAGACGGGCG

AGGTCAAGAAGGTGAAGAAGAAGCTCACCTACCAGTTCAGCGGCGAAGTGCTGG

GACGCGGGGGCCTGGCCGAGCGGCGCCACCCGCCCCACGGCAGCGGCGCCAC

AAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAAGAAAACCCCGGGCCTATG

TGTGACCGGAATGGTGGTCGGCGGCTTCGACAGTGGCTGATCGAGCAGATTGAC

AGTAGCATGTATCCAGGACTGATTTGGGAGAATGAGGAGAAGAGCATGTTCCGGA

TCCCTTGGAAACACGCTGGCAAGCAAGATTATAATCAGGAAGTGGATGCCTCCAT

TTTTAAGGCCTGGGCAGTTTTTAAAGGGAAGTTTAAAGAAGGGGACAAAGCTGAA

CCAGCCACTTGGAAGACGAGGTTACGCTGTGCTTTGAATAAGAGCCCAGATTTTG

AGGAAGTGACGGACCGGTCCCAACTGGACATTTCCGAGCCATACAAAGTTTACCG

AATTGTTCCTGAGGAAGAGCAAAAATGCAAACTAGGCGTGGCAACTGCTGGCTGC

GTGAATGAAGTTACAGAGATGGAGTGCGGTCGCTCTGAAATCGACGAGCTGATCA

AGGAGCCTTCTGTGGACGATTACATGGGGATGATCAAAAGGAGCCCTTCCCCGC

CGGAGGCCTGTCGGAGTCAGCTCCTTCCAGACTGGTGGGCGCAGCAGCCCAGC

ACAGGCGTGCCGCTGGTGACGGGGTACACCACCTACGACGCGCACCATTCAGCA

TTCTCCCAGATGGTGATCAGCTTCTACTATGGGGGCAAGCTGGTGGGCCAGGCC

ACCACCACCTGCCCCGAGGGCTGCCGCCTGTCCCTGAGCCAGCCTGGGCTGCC

CGGCACCAAGCTGTATGGGCCCGAGGGCCTGGAGCTGGTGCGCTTCCCGCCGG

CCGACGCCATCCCCAGCGAGCGACAGAGGCAGGTGACGCGGAAGCTGTTCGGG

CACCTGGAGCGCGGGGTGCTGCTGCACAGCAGCCGGCAGGGCGTGTTCGTCAA GCGGCTGTGCCAGGGCCGCGTGTTCTGCAGCGGCAACGCCGTGGTGTGCAAAG

GCAGGCCCAACAAGCTGGAGCGTGATGAGGTGGTCCAGGTCTTCGACACCAGCC

AGTTCTTCCGAGAGCTGCAGCAGTTCTATAACAGCCAGGGCCGGCTTCCTGACG

GCAGGGTGGTGCTGTGCTTTGGGGAAGAGTTTCCGGATATGGCCCCCTTGCGCT

CCAAACTCATTCTCGTGCAGATTGAGCAGCTGTATGTCCGGCAACTGGCAGAAGA

GGCTGGGAAGAGCTGTGGAGCCGGCTCTGTGATGCAGGCCCCCGAGGAGCCGC

CGCCAGACCAGGTCTTCCGGATGTTTCCAGATATTTGTGCCTCACACCAGAGATC

ATTTTTCAGAGAAAACCAACAGATCACCGTCGGCTCCGGCGAGGGCAGGGGAAG

TCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAATGTCGCAAGGGCT

CCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCGTCGCGGCGCCCGGGAACCAG

CCGCAGCCGCAGCCGCAGCAGCAGAGCCCTGAGGATGATGACAGGAAGGTCCG

AAGGAGAGAAAAAAACCGAGTTGCTGCTCAGAGAAGTCGGAAGAAGCAGACCCA

GAAGGCTGACAAGCTCCATGAGGAATATGAGAGCCTGGAGCAAGAAAACACCAT

GCTGCGGAGAGAGATCGGGAAGCTGACAGAGGAGCTGAAGCACCTGACAGAGG

CACTGAAGGAGCACGAGAAGATGTGCCCGCTGCTGCTCTGCCCTATGAACTTTGT

GCCAGTGCCTCCCCGGCCGGACCCTGTGGCCGGCTGCTTGCCCCGATGAAATCA

ACCTCTGGATTACAAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTC

CTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCC

GTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGG

AGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACG

CAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTT

CGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCG

CTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGG

GAAGGTCTGCTGAGACTCGGGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCG

CGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCC

CGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAG

ACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGTCCTCAGGTGCAGGCTG

CCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTG

AGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATC

TGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTT

TGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAAT

GAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAA

AGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCT

TATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTT ATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTC

CTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC

SEQ. ID NO: 6 mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence

(WPREmut6) - polynucleotide sequence

AATCAACCTCTGGATTACAAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT

TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC

TTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTA

TGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGC

TGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG

GACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTT

GCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTG

TCGGGGAAGGTCTGCTGAGACTCGGGGCTGCTCGCCTGTGTTGCCACCTGGATT

CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTT

CCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC

CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

SEQ. ID NO: 7 rabbit beta-globin polyadenylation signal sequence (rbBGpA) - polynucleotide sequence

TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCT

GGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCA

TGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCA

ATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAA

TCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATAT

GCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAG

CCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT

TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATG

TTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCT CTTCTCTTATGGAGATC

SEQ. ID NO: 8 late polyadenylation signal sequence of simian virus 40 (SV40late)- polynucleotide sequence CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGA

AAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA

GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGG

GGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA

SEQ. ID NO: 9

PU.1 - polynucleotide sequence

ATGTTACAGGCGTGCAAAATGGAAGGGTTTCCCCTCGTCCCCCCTCCATCAGAAG

ACCTGGTGCCCTATGACACGGATCTATACCAACGCCAAACGCACGAGTATTACCC

CTATCTCAGCAGTGATGGGGAGAGCCATAGCGACCATTACTGGGACTTCCACCCC

CACCACGTGCACAGCGAGTTCGAGAGCTTCGCCGAGAACAACTTCACGGAGCTC

CAGAGCGTGCAGCCCCCGCAGCTGCAGCAGCTCTACCGCCACATGGAGCTGGA

GCAGATGCACGTCCTCGATACCCCCATGGTGCCACCCCATCCCAGTCTTGGCCA

CCAGGTCTCCTACCTGCCCCGGATGTGCCTCCAGTACCCATCCCTGTCCCCAGC

CCAGCCCAGCTCAGATGAGGAGGAGGGCGAGCGGCAGAGCCCCCCACTGGAGG

TGTCTGACGGCGAGGCGGATGGCCTGGAGCCCGGGCCTGGGCTCCTGCCTGGG

GAGACAGGCAGCAAGAAGAAGATCCGCCTGTACCAGTTCCTGTTGGACCTGCTC

CGCAGCGGCGACATGAAGGACAGCATCTGGTGGGTGGACAAGGACAAGGGCAC

CTTCCAGTTCTCGTCCAAGCACAAGGAGGCGCTGGCGCACCGCTGGGGCATCCA

GAAGGGCAACCGCAAGAAGATGACCTACCAGAAGATGGCGCGCGCGCTGCGCAA

CTACGGCAAGACGGGCGAGGTCAAGAAGGTGAAGAAGAAGCTCACCTACCAGTT

CAGCGGCGAAGTGCTGGGACGCGGGGGCCTGGCCGAGCGGCGCCACCCGCCC

CAO

SEQ. ID NO: 10

PU.1 - polypeptide sequence

MLQACKMEGFPLVPPPSEDLVPYDTDLYQRQTHEYYPYLSSDGESHSDHYWDFHPH

HVHSEFESFAENNFTELQSVQPPQLQQLYRHMELEQMHVLDTPMVPPHPSLGHQVS

YLPRMCLQYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLLPGETGSKK

KIRLYQFLLDLLRSGDMKDSIWWVDKDKGTFQFSSKHKEALAHRWGIQKGNRKKMTY

QKMARALRNYGKTGEVKKVKKKLTYQFSGEVLGRGGLAERRHPPH

SEQ. ID NO: 11

IRF8 - polynucleotide sequence ATGTGTGACCGGAATGGTGGTCGGCGGCTTCGACAGTGGCTGATCGAGCAGATT

GACAGTAGCATGTATCCAGGACTGATTTGGGAGAATGAGGAGAAGAGCATGTTCC

GGATCCCTTGGAAACACGCTGGCAAGCAAGATTATAATCAGGAAGTGGATGCCTC

CATTTTTAAGGCCTGGGCAGTTTTTAAAGGGAAGTTTAAAGAAGGGGACAAAGCT

GAACCAGCCACTTGGAAGACGAGGTTACGCTGTGCTTTGAATAAGAGCCCAGATT

TTGAGGAAGTGACGGACCGGTCCCAACTGGACATTTCCGAGCCATACAAAGTTTA

CCGAATTGTTCCTGAGGAAGAGCAAAAATGCAAACTAGGCGTGGCAACTGCTGGC

TGCGTGAATGAAGTTACAGAGATGGAGTGCGGTCGCTCTGAAATCGACGAGCTGA

TCAAGGAGCCTTCTGTGGACGATTACATGGGGATGATCAAAAGGAGCCCTTCCCC

GCCGGAGGCCTGTCGGAGTCAGCTCCTTCCAGACTGGTGGGCGCAGCAGCCCA

GCACAGGCGTGCCGCTGGTGACGGGGTACACCACCTACGACGCGCACCATTCAG

CATTCTCCCAGATGGTGATCAGCTTCTACTATGGGGGCAAGCTGGTGGGCCAGG

CCACCACCACCTGCCCCGAGGGCTGCCGCCTGTCCCTGAGCCAGCCTGGGCTG

CCCGGCACCAAGCTGTATGGGCCCGAGGGCCTGGAGCTGGTGCGCTTCCCGCC

GGCCGACGCCATCCCCAGCGAGCGACAGAGGCAGGTGACGCGGAAGCTGTTCG

GGCACCTGGAGCGCGGGGTGCTGCTGCACAGCAGCCGGCAGGGCGTGTTCGTC

AAGCGGCTGTGCCAGGGCCGCGTGTTCTGCAGCGGCAACGCCGTGGTGTGCAA

AGGCAGGCCCAACAAGCTGGAGCGTGATGAGGTGGTCCAGGTCTTCGACACCAG

CCAGTTCTTCCGAGAGCTGCAGCAGTTCTATAACAGCCAGGGCCGGCTTCCTGAC

GGCAGGGTGGTGCTGTGCTTTGGGGAAGAGTTTCCGGATATGGCCCCCTTGCGC

TCCAAACTCATTCTCGTGCAGATTGAGCAGCTGTATGTCCGGCAACTGGCAGAAG

AGGCTGGGAAGAGCTGTGGAGCCGGCTCTGTGATGCAGGCCCCCGAGGAGCCG

CCGCCAGACCAGGTCTTCCGGATGTTTCCAGATATTTGTGCCTCACACCAGAGAT

CATTTTTCAGAGAAAACCAACAGATCACCGTC

SEQ. ID NO: 12

IRF8 - polypeptide sequence

MCDRNGGRRLRQWLIEQIDSSMYPGLIWENEEKSMFRIPWKHAGKQDYNQEVDASIF

KAWAVFKGKFKEGDKAEPATWKTRLRCALNKSPDFEEVTDRSQLDISEPYKVYRIVP

EEEQKCKLGVATAGCVNEVTEMECGRSEIDELIKEPSVDDYMGMIKRSPSPPEACRS

QLLPDWWAQQPSTGVPLVTGYTTYDAHHSAFSQMVISFYYGGKLVGQATTTCPEGC

RLSLSQPGLPGTKLYGPEGLELVRFPPADAIPSERQRQVTRKLFGHLERGVLLHSSRQ

GVFVKRLCQGRVFCSGNAVVCKGRPNKLERDEVVQVFDTSQFFRELQQFYNSQGRL

PDGRVVLCFGEEFPDMAPLRSKLILVQIEQLYVRQLAEEAGKSCGAGSVMQAPEEPP

PDQVFRMFPDICASHQRSFFRENQQITV SEQ. ID NO: 13

BATF3 - polynucleotide sequence

ATGTCGCAAGGGCTCCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCGTCGCGGC

GCCCGGGAACCAGCCGCAGCCGCAGCCGCAGCAGCAGAGCCCTGAGGATGATG

ACAGGAAGGTCCGAAGGAGAGAAAAAAACCGAGTTGCTGCTCAGAGAAGTCGGA

AGAAGCAGACCCAGAAGGCTGACAAGCTCCATGAGGAATATGAGAGCCTGGAGC

AAGAAAACACCATGCTGCGGAGAGAGATCGGGAAGCTGACAGAGGAGCTGAAGC

ACCTGACAGAGGCACTGAAGGAGCACGAGAAGATGTGCCCGCTGCTGCTCTGCC

CTATGAACTTTGTGCCAGTGCCTCCCCGGCCGGACCCTGTGGCCGGCTGCTTGC

CCCGA

SEQ. ID NO: 14

BATF3 - polypeptide sequence

MSQGLPAAGSVLQRSVAAPGNQPQPQPQQQSPEDDDRKVRRREKNRVAAQRSRK

KQTQKADKLHEEYESLEQENTMLRREIGKLTEELKHLTEALKEHEKMCPLLLCPMNFV

PVPPRPDPVAGCLPR

SEQ. ID NO: 15

SFFV promoter - polynucleotide sequence

CTGCAGCCCCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATG

AAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTGCAGTAACGCCATTTTGCAAGG

CATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATG

AAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCG

GCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCA

AGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATC

AGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTA

ACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATA

AAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGC CCGGG

SEQ ID NOs: 16-27 are empty sequences. SEQ. ID NO: 28

P2A peptide polynucleotide sequence

GCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAAGAAAACCCCGGGC

CT

SEQ. ID NO: 29

T2A peptide polynucleotide sequence

GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA

SEQ. ID NO: 30

AT-108 : Ad5-PIB (cosmid)

SEQ. ID NO: 31

AT-110: Ad5/F35-PIB (cosmid)

SEQ. ID NO: 32

AT-111: Ad5-RGD-PIB (cosmid)

SEQ. ID NO: 33

AT-112: Ad5/3-PIB (cosmid)

SEQ. ID NO: 34

69-bp MCS sequence

AGATCTTCTAGACCCGGGAGCGGCCGCTGTCGACCTGCAGGATCCGAATTCGAT

ATCACTAGTGGTACC

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Andersson, M. Hbglund, K. Miharada, Y. Nakamura, S. Hugues, L. Greiff, M. Lindstedt, F. F. Rosa, C. F. Pires, M. Bassani-Sternberg, I. M. Svane, C.-F. Pereira, Restoring tumor immunogenicity with dendritic cell reprogramming. Sc/. Immunol. 8, eadd4817 (2023).

Andrea Ziblat, Brendan L. Horton, Emily F. Higgs, Ken Hatogai, Anna Martinez, Jason W. Shapiro, Danny E.C. Kim, YuanYuan Zha, Randy F. Sweis, Thomas F. Gajewski, Batf3⁺ DCs and the 4-1 BB/4-1BBL axis are required at the effector phase in the tumor microenvironment for PD-1/PD-L1 blockade efficacy, Cell Reports Volume 43, Issue 5, 28 May 2024, 114141.

Items

1 . One or more constructs, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1 , IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late).

2. The one or more constructs according to item 1 , wherein one of the at least two transcription factors is PU.1.

3. The one or more constructs according to any one of the preceding items, which upon expression encodes PU.1 , or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 10, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 9.

4. The one or more constructs according to any one of the preceding items, which upon expression encodes IRF8, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 12, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 11 .

5. The one or more constructs according to any one of the preceding items, which upon expression encodes BATF3, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 14, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 13.

6. The one or more constructs according to any one of the preceding items, comprising: a) one construct which upon expression encodes the transcription factors PU.1 , IRF8 and BATF3; b) one construct which upon expression encodes the transcription factors IRF8 and BATF3; c) one construct which upon expression encodes the transcription factors PU.1 and BATF3; d) one construct which upon expression encodes the transcription factors PU.1 and IRF8; e) a first construct which upon expression encodes the transcription factors IRF8 and BATF3, and a second construct which upon expression encodes the transcription factor PU.1 ; f) a first construct which upon expression encodes the transcription factor BATF3, and a second construct which upon expression encodes the transcription factors PU.1 and IRF8; g) a first construct which upon expression encodes the transcription factor IRF8, and a second construct which upon expression encodes the transcription factors PU.1 and BATF3; and/or h) a first construct which upon expression encodes the transcription factor PU.1 ; a second construct which upon expression encodes the transcription factor IRF8; and a third construct which upon expression encodes the transcription factor BATF3. The one or more constructs according to any one of the preceding items, wherein PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 9. The one or more constructs according to any one of the preceding items, wherein IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 11. The one or more constructs according to any one of the preceding items, wherein BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 13. The one or more constructs according to any one of the preceding items, wherein the at least two transcription factors are in the following sequential order from 5’ to 3’:

PU.1 , IRF8, BATF3;

PU.1 , BATF3, IRF8;

IRF8, PU.1 , BATF3;

IRF8, BATF3, PU.1;

BATF3, PU.1 , IRF8; or

BATF3, IRF8, PU.1. 11. The one or more constructs according to any one of the preceding items, wherein the sequential order of transcription factors from 5’ to 3’ is PU.1, IRF8, BATF3.

12. The one or more constructs according to any one of the preceding items, wherein the SFFV promoter comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 15, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 15, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 15.

13. The one or more constructs according to any one of the preceding items, wherein the mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 6, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 6, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 6

14. The one or more constructs according to any one of the preceding items, wherein the rabbit beta-globin polyadenylation signal sequence (rbBGpA) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO:

7, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 7, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 7.

15. The one or more constructs according to any one of the preceding items, wherein the late polyadenylation signal sequence of simian virus 40 (SV40late) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO:

8, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 8, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 8.

16. The one or more constructs according to any one of the preceding items, wherein the one or more sequences are: a) WPREmut6; b) rbBGpA; c) SV40late; d) WPREmut6 and rbBGpA; e) WPREmut6 and SV40late; or f) rbBGpA and SV40late. The one or more constructs according to any one of the preceding items, wherein said one or more constructs comprise or consist of: SFFV, PU.1 , IRF8, BATF3, WPREmut6, and rbBGpA. The one or more constructs according to any one of the preceding items, wherein said one or more constructs comprise or consist of, in sequential order from 5’ to 3’: SFFV, PU.1 , IRF8, BATF3, WPREmut6, and rbBGpA. The one or more constructs according to any one of the preceding items wherein said one or more constructs comprise or consist of, in sequential order from 5’ to 3’: SFFV, PU.1 , a 2A peptide, IRF8, a 2A peptide, BATF3, WPREmut6, and rbBGpA, such as SFFV, PU.1 , P2A, IRF8, T2A, BATF3, WPREmut6, and rbBGpA, preferably wherein the P2A and T2A peptides are encoded by polynucleotide sequences comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 28 and SEQ ID NO: 29, respectively, or variants thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 28 and SEQ ID NO: 29, respectively. The one or more constructs according to any one of the preceding items, wherein said one or more constructs consist of the polynucleotide sequence set forth in SEQ ID NO: 5, or a variant thereof having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5. One or more vectors comprising the one or more constructs according to any one of the preceding items. 22. The one or more vectors according to item 21 , wherein the one or more vectors is a viral vector.

23. The one or more vectors according to any one of items 21 to 22, wherein the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retrovirus vectors, herpes virus vectors, pox virus vectors, adeno- associated virus vectors, paramyxoviridae vectors, rabdoviral vectors, alphaviral vectors, flaviral vectors, and adeno-associated viral vectors.

24. The one or more vectors according to any one of items 21 to 23, wherein the viral vector is an adenoviral vector.

25. The one or more vectors according to item 24, wherein the adenoviral vector is selected from the group consisting of: wild-type Ad vectors, chimeric Ad vectors, and mutant Ad vectors.

26. The one or more vectors according to item 25, wherein the wild-type Ad vector is Ad5.

27. The one or more vectors according to any one of items 24 to 26, wherein the Ad vector is selected from the group consisting of: Ad5-RGD, Ad5/F35 and Ad5/3.

28. The one or more vectors according to any one of items 22 to 23, wherein the viral vector is a lentiviral vector.

29. The one or more vectors according to item 23, wherein the adeno-associated viral vector is selected from the group consisting of: wild-type AAV vectors, hybrid AAV vectors and mutant AAV vectors.

30. The one or more vectors according to item 29, wherein the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV2-QuadYF. 31. The one or more vectors according to any one of the preceding items, wherein one or more Sfil sites have been mutated, preferably by silent mutations, even more preferably wherein said Sfil sites are in the pVII ORF and/or the adenovirus DNA-binding protein (DBP) ORF of the vector.

32. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ I D NO: 1 , or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1.

33. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 2.

34. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 3.

35. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 4.

36. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 30, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 30.

37. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ I D NO: 31 , or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 31.

38. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 32, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 32.

39. The one or more vectors according to any one of the preceding items, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 33, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 33.

40. A method of manufacturing the one or more vectors according to any one of the preceding items, said method comprising: a) Providing a host cell capable of being transfected with a nucleic acid sequence encoding the one or more vectors, such the adenoviral vectors according to any one of items 23 to 39; b) Transfecting the host cell with the nucleic acid sequence encoding the one or more vector; c) Culturing the transfected host cell under conditions suitable for expression and assembly of the one or more vector, such as adenoviral particles; d) Harvesting the vector from the cultured host cells; and e) Purifying the harvested vector. A cell comprising the one or more constructs or the one or more vectors according to any one of items 1 to 39. The cell according to item 41 , wherein the cell comprises: a) one construct or vector which upon expression encodes the transcription factors PU.1 , IRF8 and BATF3; b) one construct or vector which upon expression encodes the transcription factors IRF8 and BATF3; c) one construct or vector which upon expression encodes the transcription factors PU.1 and BATF3; d) one construct or vector which upon expression encodes the transcription factors PU.1 and IRF8; e) a first construct or vector which upon expression encodes the transcription factors IRF8 and BATF3, and a second construct or vector which upon expression encodes the transcription factor PU.1 ; f) a first construct or vector which upon expression encodes the transcription factor BATF3, and a second construct or vector which upon expression encodes the transcription factors PU.1 and IRF8; g) a first construct or vector which upon expression encodes the transcription factor IRF8, and a second construct or vector which upon expression encodes the transcription factors PU.1 and BATF3; and/or h) a first construct or vector which upon expression encodes the transcription factor PU.1; a second construct or vector which upon expression encodes the transcription factor IRF8; and a third construct or vector which upon expression encodes the transcription factor BATF3. The cell according to any one of the preceding items, wherein the transcription factors are as defined in the constructs of any one of items 1 to 18. The cell according to any one of the preceding items, wherein the one or more constructs are as defined in any one of items 1 to 26. 45. The cell according to any one of the preceding items, wherein the one or more vectors are as defined in any one of items 27 to 45.

46. The cell according to any one of the preceding items, wherein the cell is a mammalian cell.

47. The cell according to any one of the preceding items, wherein the cell is a human cell.

48. The cell according to any one of the preceding items, wherein the cell is a murine cell.

49. The cell according to any one of the preceding items, wherein the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell.

50. The cell according to item 49, wherein the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a hematopoietic stem cell.

51. The cell according to item 49, wherein the differentiated cell is any somatic cell.

52. The cell according to item 51 , wherein the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte.

53. The cell according to any one of the preceding items, wherein the cell is expressing one or more of the surface markers selected from the group consisting of: Coxsackie and Adenovirus Receptor (CAR), CD51 , CD46 and DSG-2.

54. A method for reprogramming or inducing a cell into a dendritic cell or antigen- presenting cell, comprising the following step: a) transducing a cell with the one or more constructs of any one of items 1 to 20 or the one or more vectors according to any one of items 21 to 39. A method for reprogramming or inducing a cell into a dendritic cell or antigen- presenting cell, comprising the following steps: a) transducing a cell with the one or more constructs of any one of items 1 to 21 or the one or more vectors according to any one of items 21 to 39; b) expressing the transcription factors whereby a reprogrammed or induced cell is obtained. The method according to any one of items 54 and 55, wherein the method further comprises culturing the transduced cell in a media comprising one or more cytokines. The method according to item 56, wherein the one or more cytokines are pro- inflammatory cytokines. The method according to any one of items 56 to 57, wherein the one or more cytokines are hematopoietic cytokines. The method according to any one of items 56 to 58, wherein the one or more cytokines are selected from the group consisting of: I FNfB, IFNy, TNFa, IFNa, IL-1 , IL-6, CD40I, Flt3l, GM-CSF, IFN-A1 , IFN-w, IL-2, IL-4, IL-15, prostaglandin 2, SCF and oncostatin M (OM). The method according to any one of items 56 to 59, wherein the one or more cytokines are selected from the group consisting of: I FN|3, IFNy and TNFa. The method according to any one of items 54 to 60, wherein the transducing step further comprises at least one vector comprising nucleic acids encoding immunostimulatory cytokines and/or siRNA targeting immunosuppressive cytokines, including but not restricted to IL-10. The method according to any one of items 54 to 61 , wherein the cell is a mammalian cell. The method according to any one of items 54 to 62, wherein the cell is a human cell. 64. The method according to any one of items 54 to 63, wherein the cell is a murine cell.

65. The method according to any one of items 54 to 64, wherein the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell.

66. The method according to item 65, wherein the cancer cell is selected from the group consisting of: a brain cancer cell, such as glioblastoma, a melanoma cancer cell, an ovarian cancer cell, and a gastrointestinal cancer cell, such as a gastric carcinoma cancer cell.

67. The method according to item 65, wherein the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a hematopoietic stem cell.

68. The method according to item 65, wherein the differentiated cell is any somatic cell.

69. The method according to item 68, wherein the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte.

70. The method according to any one of items 54 to 69, wherein the cell is expressing one or more of the surface markers selected from the group consisting of: Coxsackie and Adenovirus Receptor (CAR), CD51 , CD46 and DSG-2.

71. The method according to any one of items 54 to 70, wherein the transduced cell is cultured during at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

72. The method according to any one of items 54 to 71 , wherein the resulting reprogrammed or induced cell is a type 1 conventional dendritic cell. The method according to any one of items 54 to 72, wherein the resulting reprogrammed or induced cell is cluster differentiation 45 (CD45) positive. The method according to any one of items 54 to 73, wherein the resulting reprogrammed or induced cell is human leukocyte antigen-DR isotype (HLA- DR) positive. The method according to any one of the preceding items, wherein the multiplicities of infection (MOI) is 1 to 2000, such as 5 to 1500, such as 10 to 1000, such as 100 to 1000, such as 200 to 1000, such as 300 to 1000, such as 400 to 1000, such as 500 to 1000. The method according to any one of the preceding items, wherein the multiplicities of infection (MOI) is 1 to 100, such as 10 to 100, such as 20 to 100, such as 30 to 100, such as 40 to 100, such as 50 to 100, such as 60 to 100, such as 70 to 100, such as 80 to 100, such as 90 to 100. The method according to any one of the preceding items, wherein the multiplicities of infection (MOI) is 50 to 500, such as 50 to 400, such as 50 to 300, such as 50 to 200, such as 100 to 200. The method according to any one of items 54 to 77, wherein day 0 is the day of the transduction step and wherein the step of culturing the transduced cell is performed from day 0 to day 9. A reprogrammed or induced cell obtained by the method defined in any one of items 54 to 78. The reprogrammed or induced cell according to item 79, wherein the cell is a dendritic or antigen-presenting cell, such as a type 1 conventional dendritic cell. The reprogrammed or induced cell according to any one of items 79 to 80, wherein the cell is cluster differentiation 45 (CD45) positive. The reprogrammed or induced cell according to any one of items 79 to 81 , wherein the cell is cluster differentiation 226 (CD226) positive. The reprogrammed or induced cell according to any one of items 79 to 82, wherein the cell is human leukocyte antigen DR isotype (H LA-DR) positive. The reprogrammed or induced cell according to any one of items 79 to 83, wherein the resulting reprogrammed or induced cell is CD45, HLA-DR, CD141 , CLEC9A, XCR1 and/or CD226 positive. A pharmaceutical composition comprising the one or more constructs, the one or more vectors, the cells, or the reprogrammed or induced cells according to any one of the preceding items, and a pharmaceutically acceptable carrier, diluent, or excipient. The one or more constructs according to any one of items 1 to 20, the one or more vectors according to any one of items 21 to 39, the cell according to any one of items 41 to 53, the reprogrammed or induced cell according to any one of items 79 to 84, and/or the pharmaceutical composition according to item 85, for use in medicine. The one or more constructs according to any one of items 1 to 20, the one or more vectors according to any one of items 21 to 39, the cell according to any one of items 41 to 53, the reprogrammed or induced cell according to any one of items 79 to 84, and/or the pharmaceutical composition according to item 85 for use as a medicament. The one or more constructs according to any one of items 1 to 20, the one or more vectors according to any one of items 21 to 39, the cell according to any one of items 41 to 53, the reprogrammed or induced cell according to any one of items 79 to 84, and/or the pharmaceutical composition according to item 85 for use in the treatment of cancer, such as solid tumor cancers and/or hematological cancers. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to item 88, wherein the cancer is selected from the group consisting of: colorectal cancer, head and neck cancer, melanoma, breast cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, a germ cell tumor, a retinoblastoma, an age-related macular degeneration, glioblastoma, lymphoma, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, oesophageal cancer, uterus cancer, vaginal cancer, gastric cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, bladder and urothelial cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, skin cancer, and myeloma. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 88 to 89, wherein the cancer is selected from the group consisting of: melanoma, breast cancer, head and neck cancer, such as head and neck squamous cell carcinoma (HNSCC), such as head and neck squamous cell carcinoma (HNSCC) with combined positive score of PD-L1 less than 1 , sarcoma, colorectal cancer, such as metastatic microsatellite stable colorectal cancer. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 88 to 89, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, head and neck cancer, colorectal cancer, sarcoma, liver cancer, and ovarian cancer. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 88 to 89, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, and head and neck cancer.

93. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 88 to 89, wherein the cancer is selected from the group consisting of: glioblastoma, melanoma, sarcoma, head and neck cancer, and melanoma.

94. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to item 93, wherein the cancer is selected from the group consisting of: glioblastoma, melanoma, sarcoma, and head and neck cancer.

95. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 93 to 94, wherein the cancer is selected from the group consisting of: melanoma, and head and neck cancer.

96. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to item 94, wherein the cancer is selected from the group consisting of: melanoma and sarcoma.

97. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to item 94, wherein the cancer is selected from the group consisting of: melanoma and glioblastoma.

98. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of the preceding items, wherein the cancer is a cancer having a low tumor mutational burden (TMB), preferably, wherein the low TMB is characterized by at the most 2000 non-synonymous mutations, such as at the most 1500 non-synonymous mutations, such as a the most 1000 non- synonymous mutations, even more preferably wherein said low TMB is measured using whole-exome sequencing (WES) data.

99. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 98, as a monotherapy or in combination with other anti-cancer therapeutic(s), such as immunotherapy, such as immune checkpoint blockade inhibitor(s), preferably anti-PD1, anti-PD-L1 , or anti-CTLA4 therapeutic(s).

100. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 99, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition, and the other anti-cancer therapeutic(s) are administered simultaneously, sequentially or separately.

101. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 100, wherein the cancer is resistant to immune checkpoint blockade inhibition therapy.

102. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 101 , wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition are administered intratumorally or systemically, such as intravenously, intraperitoneally, or subcutaneously.

103. The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 102, further comprising measuring the number of intratumoral CD8+ T cells, CD4+ T cells, NK cells and/or B cells. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 103, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition increase the number of intratumoral lymphocytes, such as CD8+ T cells, CD4+ T cells, NK cells and/or B cells, preferably the number of intratumoral CD8+ T cells. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 104, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition increase the number of effector and/or central memory T cells, such as CD44+CD62L+ and CD44+CD62L- T cells, respectively. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 105, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition increase the proliferation of CD8+ T cells and/or CD4+ T cells. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 106, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition increase the number of TCF-1+CD8+ T cells and/or the number of TCF-1+CD4+ T cells. . The one or more constructs the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of the preceding items, wherein the number of tumor infiltrating lymphocyte (TIL) B cell populations, such as lgM⁺ B cells (CD19+ B220+ lgM+), activated B cells (CD19+ B220+ CD95+ GL7+), plasma cells (CD45+ CD19- CD138+), and/or follicular dendritic cells (DCs) (CD45+ CD19- CD23+) is increased. . The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the production of circulating tumor-specific IgM, lgG1, and/or IgA antibodies is activated. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 109, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition reduces T cell exhaustion, preferably intratumoral T cell exhaustion. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 110, further comprising measuring the presence of tertiary lymphoid structures (TLS), such as intratumoral tertiary lymphoid structures (TLS), such as tertiary lymphoid structures (TLS) in the tumor parenchyma. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to item 111 , wherein the presence of tertiary lymphoid structures (TLS) is performed based on podoplanin expression, such as podoplanin immunostaining. . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 112, wherein the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition leads to the formation of tertiary lymphoid structures (TLS), or increases the formation of tertiary lymphoid structures (TLS). . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 113, wherein administration of the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition leads to at least one effect selected from the group consisting of: delay of tumor growth, inhibition of tumor growth, tumor regression, reduced metastasis, abscopal effect, increase in overall survival, complete response, partial response . The one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition for use according to any one of items 86 to 114, wherein the increase or the at least one effect is observed after at least 1 , such as at least 2, such as at least 3, such as at least 4 administrations of the one or more constructs, the one or more vectors, the cell, the reprogrammed or induced cell, and/or the pharmaceutical composition. . The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are administered to an individual in need thereof 1 time, 2 times, 3 times, or 4 times. . The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are administered to an individual in need thereof at least 3 times, such as at least 4 times, such as at least 5 times. . The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the immune checkpoint inhibitors are administered to the individual in need thereof at least 3 times, such as at least 4 times, such as at least 5 times.

119. The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the immune checkpoint inhibitors are administered simultaneously, sequentially, and/or intercalated with the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell.

120. The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein a first lead cycle of administrations of the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell is performed, followed by a second cycle of administrations.

121. The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the lead cycle comprises 3 administrations, and the second cycle 2 administrations.

122. The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the lead cycle administrations are performed at days 0, 2 and 4, and the second cycle administrations are performed at days 9 and 14, wherein day 0 is the day of the first administration.

123. The one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell for use according to any one of the preceding items, wherein the lead cycle administrations are performed at days 0, 2 and 4, and the second cycle of administrations are performed at days 9 and 14, and immune checkpoint inhibitors are administered at days 0, 3, and 7, wherein day 0 is the day of the first administration. . A method of treating cancer, the method comprising administering to an individual in need thereof the one or more constructs according to any one of items 1 to 20, the one or more vectors according to any one of items 21 to 39, the cell according to any one of items 41 to 53, the reprogrammed or induced cell according to any one of items 79 to 84, and/or the pharmaceutical composition according to item 85. . Use of the one or more constructs according to any one of items 1 to 20, the one or more vectors according to any one of items 21 to 39, the cell according to any one of items 41 to 53, the reprogrammed or induced cell according to any one of items 79 to 84, and/or the pharmaceutical composition according to item 85 for the manufacture of a medicament for the treatment of cancer. . A method for determining efficacy of cancer treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell according to any one of the preceding items, the method comprising: determining the levels of tumor infiltrating lymphocyte (TIL) B cell populations, such as lgM⁺ B cells (CD19+ B220+ lgM+), activated B cells (CD19+ B220+ CD95+ GL7+), plasma cells (CD45+ CD19- CD138+), and/or follicular dendritic cells (DCs) (CD45+ CD19- CD23+) in a first a biological sample of an individual having received the treatment; comparing the levels of the TILs with the levels of the TILs in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said TILs in the first sample compared to the second sample indicates efficacious treatment. 127. The method according to item 126, wherein the biological sample is a tumor biopsy.

128. The method according to any one of items 126 and 127, wherein the step of determining is performed by tumor immunophenotyping.

129. A method for determining efficacy of cancer treatment using the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell according to any one of the preceding items, the method comprising: determining the levels of tumor-specific IgM, lgG1 , and/or IgA antibodies in a first a biological sample of an individual having received the treatment; comparing the levels of the tumor-specific IgM, IgG 1 , and/or IgA antibodies with the levels of the tumor-specific IgM, lgG1 , and/or IgA antibodies in a sample obtained from said individual before, or earlier in the treatment of said individual, wherein an increase in the number of said tumor-specific IgM, lgG1, and/or IgA antibodies in the first sample compared to the second sample indicates efficacious treatment.

130. The method according to item 129, wherein the biological sample is a blood sample or a serum sample.

131. The method according to any one of items 129 and 130, wherein the step of determining is performed by tumor-specific antibody binding.

132. The method according to any one of items 126 to 131 , wherein the one or more constructs, the one or more vectors, the cell, the pharmaceutical composition, and/or the reprogrammed or induced cell are for use according to any one of the preceding items.

133. The method according to any one of items 126 to 132, wherein the cancer is selected from the group consisting of: colorectal cancer, head and neck cancer, melanoma, breast cancer, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, a germ cell tumor, a retinoblastoma, an age-related macular degeneration, glioblastoma, lymphoma, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, oesophageal cancer, uterus cancer, vaginal cancer, gastric cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, bladder and urothelial cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, skin cancer, and myeloma.

Claims

2. The one or more constructs according to claim 1, wherein: a) the mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 6, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 6, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 6; b) the rabbit beta-globin polyadenylation signal sequence (rbBGpA) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 7, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 7, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 7; and/or c) the late polyadenylation signal sequence of simian virus 40 (SV40late) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 8, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 8, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 8.

3. The one or more constructs according to any one of the preceding claims, wherein the one or more sequences are: a) WPREmut6; b) rbBGpA; c) SV40late; d) WPREmut6 and rbBGpA; e) WPREmut6 and SV40late; or f) rbBGpA and SV40late.

4. The one or more constructs according to any one of the preceding claims, wherein: a) PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 9, b) IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 11 , and/or c) BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 13.

5. The one or more constructs according to any one of the preceding claims, wherein said one or more constructs comprise or consist of: SFFV, PU.1 , IRF8, BATF3, WPREmut6, and rbBGpA.

6. The one or more constructs according to any one of the preceding claims, wherein said one or more constructs comprise or consist of, in sequential order from 5’ to 3’: SFFV, PU.1 , P2A, IRF8, T2A, BATF3, WPREmut6, and rbBGpA, optionally wherein the P2A and T2A peptides are encoded by polynucleotide sequences comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 28 and SEQ ID NO: 29, respectively, or variants thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 28 and SEQ ID NO: 29, respectively.

7. The one or more constructs according to any one of the preceding claims, wherein said one or more constructs consist of the polynucleotide sequence set forth in SEQ ID NO: 5, or a variant thereof having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5.

8. One or more vectors comprising the one or more constructs according to any one of the preceding claims.

9. The one or more vectors according to claim 8, wherein the vector is a viral vector.

10. The one or more vectors according to claim 9, wherein the viral vector is an adenoviral vector (Ad), preferably wherein the Ad vector is selected from the group consisting of: Ad5-RGD, Ad5/F35 and Ad5/3.

11. The one or more vectors according to any one of the preceding claims, wherein the vector is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 1 , or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1.

12. A method of manufacturing the one or more vectors according to any one of claims 8 to 11, said method comprising: a) Providing a host cell capable of being transfected with a nucleic acid sequence encoding the one or more vectors, such the adenoviral vector according to claim 10; b) Transfecting the host cell with the nucleic acid sequence encoding the one or more vectors; c) Culturing the transfected host cell under conditions suitable for expression and assembly of the vector, such as adenoviral particles; d) Harvesting the vector from the cultured host cells; and e) Purifying the harvested vectors.

13. A cell comprising the one or more constructs of any one of claims 1 to 7 or the one or more vectors according to any one of claims 8 to 11 .

14. A method for reprogramming or inducing a cell into a dendritic cell or antigen- presenting cell, comprising the step of transducing a cell with the one or more constructs of any one of claims 1 to 7 or the one or more vectors according to any one of claims 8 to 11 .

15. A reprogrammed or induced cell obtained by the method of claim 14.

16. A pharmaceutical composition comprising the one or more constructs according to any one of claims 1 to 7, the one or more vectors according to any one of claims 8 to 11, the cell according to any claim 13, or the reprogrammed or induced cell according to claim 15

17. The one or more constructs according to any one of claims 1 to 7, the one or more vectors according to any one of claims 8 to 11 , the cell according to any claim 13, or the reprogrammed or induced cell according to claim 15, for use as a medicament.

18. The one or more constructs according to any one of claims 1 to 7, the one or more vectors according to any one of claims 8 to 11 , the cell according to any claim 13, or the reprogrammed or induced cell according to claim 15, for use in the treatment of cancer, such as solid tumor cancers and/or hematological cancers.

19. The one or more constructs, the one or more vectors, the cell, or the reprogrammed or induced cell for use in the treatment of cancer according to claim 18, wherein the cancer is selected from the group consisting of: melanoma, lung cancer, breast cancer, head and neck cancer, colorectal cancer, sarcoma, liver cancer, and ovarian cancer.