Cancer cells and embryonic tissues share a number of cellular and molecular properties, suggesting that induced pluripotent stem cells (iPSCs) may be harnessed to elicit anti-tumor responses in cancer vaccines. RNA sequencing revealed that human and murine iPSCs express tumor-associated antigens, and we show here a proof of principle for using irradiated iPSCs in autologous anti-tumor vaccines. In a prophylactic setting, iPSC vaccines prevent tumor growth in syngeneic murine breast cancer, mesothelioma, and melanoma models. As an adjuvant, the iPSC vaccine inhibited melanoma recurrence at the resection site and reduced metastatic tumor load, which was associated with fewer Th17 cells and increased CD11b + GR1 hi myeloid cells. Adoptive transfer of T cells isolated from vaccine-treated tumor-bearing mice inhibited tumor growth in unvaccinated recipients, indicating that the iPSC vaccine promotes an antigen-specific anti-tumor T cell response. Our data suggest an easy, generalizable strategy for multiple types of cancer that could prove highly valuable in clinical immunotherapy.

Since the discovery of induced pluripotent stem cells (iPSCs) (), pluripotent cells from a patient’s own tissues can be created that share nearly identical gene expression and surface markers profiles with ESCs (), circumventing a major ethical roadblock. Additionally, the tumorigenic () and immunogenic () properties of iPSCs with autologous transplantation suggest potential efficacy in cancer vaccination. Importantly, autologous iPSCs may provide a more accurate and representative panel of patient’s tumor immunogens than non-autologously derived ESCs. Here, we test the hypothesis that iPSCs may work as a whole-cell-based vaccine that presents the immune system with a broad heterogeneity in cancer-related epitopes.

Nearly a century ago, researchers observed that immunization with embryonic materials led to the rejection of transplanted tumors (). More recently, studies identified shared transcriptome profiles and antigens on various tumor cells and embryonic cells (). This has led to the hypothesis that embryonic stem cells (ESCs) could be used as immunization agents to promote an anti-tumor response. A major advantage of whole-cell vaccination over traditional vaccines, which consist of inactivated organisms or protein products, is that a broad range of antigens can be presented to T cells, including unknown antigens (). However, the use of fetal and embryonic materials as vaccines to induce anti-tumor immunity has not yet advanced beyond animal models, owing largely to ethical challenges surrounding these therapies.

To assess the effectiveness of the vaccine as an adjuvant therapy after tumor resection, we next injected 5 × 10B16F0 tumor cells subcutaneously in the lower back of C57BL/6 mice and R2- or R1-resected the tumors after 2 weeks. R2-resected mice had no visible recurrence of melanoma in the resection area (RA) after receiving two adjuvant rounds of C+I vaccine, whereas PBS-control-vaccinated mice had visible tumors within the RAs ( Figure S7 A). R1-resected mice were vaccinated for 4 weeks with the C+I vaccine (n = 10), CpG (n = 10), and PBS (n = 8) ( Figure 7 A), after which dLNs and RAs were analyzed using a tumor-specific primer designed to detect and quantify the B16F0 melanoma line ( Figures S7 B–S7G). Tumor load in the dLNs was reduced in both CpG-only and the C+I-vaccine groups, indicating that CpG acted as a potent adjuvant to induce tumor degradation upon near-tumor injection ( Figure S7 H). Interestingly, in areas more distant from the vaccination sites, only the C+I-vaccinated group had significantly lower tumor recurrence in the RA ( Figure 7 B). Systemically, this is explained by reactivation of the immune system (), as well as a reduction of B16 melanoma-promoting Th17 cells () compared to the control groups ( Figures 7 C, S5 C, and S5E).

(B) DNA from skin biopsy specimens ( ∗ ) in resection areas (RAs) showed a significant reduction in the percentage of tumor cells after four vaccination rounds with the C+I vaccine, as assessed by ddPCR.

As an alternative model for prophylactic treatment, we selected the mesothelioma cell line AC29, syngeneic to CBA/J mice. Again, CBA/J iPSCs were created ( Figures S2 C and S2D), and mice were vaccinated for 4 weeks with PBS (P), CpG and iPSCs (C+I), or CpG with irradiated AC29 cancer cells (C+A) as a positive control. Afterward, 2 × 10AC29 cells (A) or 2 × 10iPSCs (I) were injected subcutaneously, and after 1 week, the TILs were analyzed for their immune profile and T cell receptor (TCR) sequences. Immune profiling was performed with cytometry by time-of-flight (CyTOF) analysis using a phenotype and intracellular staining kit, which revealed an increased presence of effector/memory CD4(24.0%) and CD8T cells (22.4%), with a reduction in T-regs in the C+I/A group (1.9%) compared to P/A control (21.1%, 14.2%, and 3.0%, respectively) ( Figure 5 A). Using Citrus (cluster identification, characterization, and regression) analysis (), B cells and T cells expressing interleukin-2 (IL-2), IL-4, and IL-5 were found to be predictive of tumor regression in C+I-vaccinated mice compared to the PBS control group ( Figures 5 B, S5 A, S5B, and S5D). Interestingly, systemic cytokine levels were significantly lower in the vaccinated group and were found to correlate with the positive control mice showing iPSC and tumor rejection (C+I/iPSC; C+A/AC29, respectively) ( Figures 6 A, S6 A, and S6B). TCR sequencing in the PBS control group revealed an overlap in T cell clones that are commonly present in thymus and spleen ( Figure S6 C). In contrast, the TCRs in the C+I group were more diverse among different mice. In addition, there was a generally lower frequency of the clones in the thymus and more similar frequencies in the spleen, likely because of mouse-specific responses to the C+I vaccine ( Figures 6 B and S6 D). Interestingly, there was one TCR clone that was shared by four of five mice in the C+I group but was not present in any of the other groups; this clone was also extremely rare in naive mice.

(B) Among C+I-vaccinated mice (C+I1 through C+I5/AC29), there was greater unique vaccine-associated variance within the TILs, whereas PBS-vaccinated mice (PBS1 through 5/AC29) demonstrated a higher uniformity among T cells that are commonly present in lymphoid organs ( Figures S6 C and S6D).

(A) Luminex analysis of serum from the different treatment groups 1 week after tumor cell introduction reveals a significantly lower presence of systemic cytokines in the positive control mice (C+I/iPSC, C+A/AC29) compared to PBS control mice (PBS/AC29). The C+I/AC29 group follows a similar trend as the positive control samples (C+I/iPSC and C+A/AC29; ANOVA with Tukey’s multiple comparison test; ∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗ p < 0.001).

(B) Citrus analysis of CyTOF data revealed that higher levels of IL-2, IL-4, and IL-5 in B cell and helper T cell clusters in the C+I mice are responsible for the intra-tumoral immune response.

(A) 1 week after 2 × 10 6 AC29 (A) mesothelioma cells were injected in CpG+iPSC (C+I)-vaccinated mice (n = 5), TILs in this C+I/A group showed an increase in the frequency of effector/memory CD4 + and CD8 + cells and a reduction in T-reg numbers compared to PBS (P)-vaccinated mice (n = 5; P/A group), as assessed by spanning tree progression analysis of density-normalized events (SPADE) of CyTOF data. The positive control groups, C+I-vaccinated and CpG+AC29 (C+A)-vaccinated mice, fully rejected iPSCs (n = 5; C+I/I) and AC29 cells (n = 5; C+A/A), respectively, with a subsequently enhanced presence of monocytes and macrophages and stromal cells.

To test whether the C+I vaccine provides immunity against shared epitopes between iPSCs and cancer cells, we performed additional experiments to assess two-way immunity by demonstrating (1) cancer immunity by C+I primed T cells and (2) iPSC immunity by tumor-experienced lymphocytes (TELs). For the first experiment, isolated T cells from C+I-vaccinated or vehicle (PBS+CpG)-vaccinated mice were adoptively transferred to a group of tumor-bearing orthotopic breast cancer mice (n = 7 per group), and tumor growth was measured over the course of 4 weeks ( Figure 4 A). This resulted in a significant reduction of tumor sizes in the C+I-vaccinated group compared to the vehicle-vaccinated group as early as 1 week after the adoptive transfer ( Figure 4 B). For the second experiment, another batch of mice vaccinated with C+I (n = 10) or vehicle (n = 10) were inoculated with breast cancer cells, and tumor growth was measured at 1 week ( Figures 4 C and 4D). Afterward, we extracted TELs from the dLNs near the tumor site (). These TELs were then adoptively transferred to iPSC-inoculated non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice (5 × 10TELs per mouse; n = 4 per group), and teratoma development was measured for 4 weeks. Significant reduction in teratoma sizes was seen at 4 weeks in the NOD-SCID mice receiving TELs from C+I animals that were able to reject the DB7 tumor cells, whereas mice receiving TELs from vehicle-vaccinated animals developed large teratomas ( Figure 4 E and 4F).

(E) Representative images of NOD-SCID mice receiving TELs from the dLNs from vehicle- or C+I-vaccinated mice from the experiment in (C) (n = 4 per group).

(B) Quantification of the tumor sizes of tumor-bearing mice in (A) over the course of 4 weeks after receiving T cells from vehicle- or C+I-vaccinated mice, as measured by caliper. Significant reduction of tumor sizes was seen as early as 1 week after the adoptive transfer of T cells from C+I-vaccinated mice and remained significantly reduced during the course of the experiment ( ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student t test).

At 4 weeks, FVB mice in the C+I-vaccinated group had significant increases in the effector/memory cytotoxic T cells in the spleen and dLNs ( Figures 2 D and 2F). The tumor specificity of these cytotoxic T cells was further confirmed by increased secretion of interferon-γ (IFN-γ) by splenocytes isolated from C+I-vaccinated mice in response to DB7 tumor lysate ( Figures 3 A, 3B, S4 A, and S4B). As with the C57BL/6 mice, upregulation of mature APCs and helper T cells was also seen in dLNs of FVB mice ( Figures 2 E and 2F). Both mouse strains remained healthy throughout the study and showed no signs of autoimmune responses due to the vaccine in serum and in tissues ( Figures S4 C–S4F). Lastly, the effectiveness of the C+I vaccine was assessed in the more clinically relevant orthotopic model of breast cancer. Significant tumor size differences were seen as early as 1 week after orthotopic transfer of cancer cells in C+I-vaccinated mice compared to vehicle control, followed by further tumor reduction over the course of 3 weeks ( Figures 3 C and 3E). Using an additional group of orthotopic breast cancer mice, in vivo tumor specificity was tested by adoptively transferring splenocytes from C+I vaccinated or vehicle (PBS+CpG) vaccinated mice into these tumor-bearing mice ( Figure 3 D). This resulted in a significant reduction of tumor sizes in the C+I-vaccinated group compared to the vehicle-vaccinated group ( Figure 3 F).

(F) Significant reduction of tumor volume in tumor-bearing mice from (D) over the course of 3 weeks after adoptive transfer of splenocytes from C+I-vaccinated mice (n = 7) compared to mice receiving splenocytes from vehicle-vaccinated mice (n = 8). ∗∗∗ p < 0.001 (one way ANOVA).

(E) Quantification of the results from (C) shows a significant reduction of tumor volume in C+I-vaccinated mice compared to vehicle-vaccinated mice in an orthotopic tumor model of breast cancer over the course of 3 weeks. ∗∗∗ p < 0.001 (one way ANOVA).

(D) Representative images of tumor volume in tumor bearing mice after receiving adoptive transfer of splenocytes from C+I-vaccinated mice compared to vehicle-vaccinated mice in an orthotopic tumor model of breast cancer 3 weeks after adoptive transfer.

(B) Significant increase of number of IFN-γ spots in the C+I-vaccinated group compared to the vehicle group. Spots were calculated using Adobe Photoshop software based on color differences. ∗∗∗ p < 0.001 (Student’s t test).

(A) Dual ELISPOT assay (red, granzyme-β; blue, IFN-γ) for immune cell activation of splenocytes in the C+I-vaccinated group (n = 6) compared to CpG alone (vehicle; n = 4) group upon exposure to iPSC lysate and DB7 lysate (see also Figures S4 A and S4B).

To test the effectiveness of our vaccine in targeting multiple cancer types, an additional experiment was performed using the melanoma cell line B16F0, which is syngeneic to the C57BL/6 mouse strain. C57BL/6 iPSCs were generated ( Figures S2 B and S2D), and 40 mice were again divided into PBS, CpG, iPSCs, and C+I groups and treated for 4 weeks. Following this, 5 × 10B16F0 cells were subcutaneously injected in the lower back. Tumor growth assessment by caliper measurement showed significantly lower tumor progression by week 2 in the C+I group ( Figures 1 E, 1F, S3 E, and S3F). Due to large tumor sizes in the control groups, the mice were sacrificed 2 weeks after tumor injection. Afterward, the immune cell profiles in blood, dLNs, and spleens were analyzed using flow cytometry. Cytometric analysis showed a significant decrease in CD4CD25FoxP3regulatory T cells (T-regs) in blood and an increase in effector/memory helper T cells in dLNs 2 weeks after tumor injections in C57BL/6 mice ( Figures 2 A and 2B ), as well as increased percentages of mature antigen-presenting cells (APCs) ( Figure 2 C).

(E and F) dLNs of these mice revealed an increased frequency of mature antigen-presenting macrophages (E) as well as effector/memory helper T cells and cytotoxic T cells (F).

(B and C) The dLNs in the C+I group had significantly higher percentages of effector/memory helper T cells (B) and increased antigen presentation by mature antigen-presenting cells (APCs) such as macrophages (CD11b + F4/80 + MHC-II + CD86 + ) and dendritic cells (CD11c + MHC-II + CD86 + ) (C).

(A) 2 weeks after B16F0 introduction, iPSC- and C+I-vaccinated C57BL/6 mice showed a significant reduction in percentages of regulatory T cells (CD4 + CD25 + FoxP3 + ) and an increase in effector/memory helper T cells (CD4 + CD44 + ) in the peripheral blood of C+I-vaccinated mice. At that point, only limited upregulation of effector/memory cytotoxic T cells (CD8 + CD44 + ) was seen.

Using FVB strain iPSCs ( Figures S2 A and S2D) and the adjuvant CpG, proven to be successful in tumor vaccination (), we observed an effective immune response to a murine breast cancer (DB7) with a CpG and iPSCs (C+I) combination. In brief, we first established the effect of CpG and an optimal vaccination schedule. We primed FVB mice with iPSCs or C+I for 2 weeks or 4 weeks and found the strongest in vitro T cell responses to DB7 tumor lysate in the C+I 4-week group ( Figures S2 E and S2F). In addition, a vaccination schedule of 4 weeks with the C+I combination resulted in the highest immunoglobulin G (IgG) binding (80.0% ± 3.4%) to DB7 and was therefore used for subsequent vaccination rounds ( Figures 1 A and 1B ). After optimizing the schedule ( Figure 1 C), we proceeded with the vaccination of 40 FVB mice divided into four groups: (1) PBS, (2) CpG only, (3) iPSCs only, and (4) C+I. After four once-weekly vaccinations, 5 × 10DB7 cancer cells were injected subcutaneously, and tumor size was monitored using caliper measurement. After 1 week, all mice presented with a similar lesion at the injection site that regressed in 7 out of 10 C+I-treated mice and progressed to larger tumors in the other groups ( Figures 1 D, S3 A, and S3B). Four weeks after tumor inoculation, five mice per group were sacrificed to analyze the immune profiles in blood, spleen, and draining lymph nodes (dLNs). The other five mice per group were used for long-term survival studies for up to 1 year. Most were sacrificed in the first 2 weeks after the end of the experiment when their tumor exceeded 1 cm. However, two mice in the C+I treatment group survived 1 year and had antibody titers against iPSCs and DB7 similar to the start of the experiment and were able to fully reject 5 × 10cancer cells upon reintroduction ( Figures S3 C and S3D). The control mice in this experiment, primed with iPSC-derived endothelial cells, were unable to mount IgG responses to the DB7 cell line, thereby ruling out the possibility that the culturing conditions with FBS-containing media could be responsible for the cross-reactivity or endogenous murine leukemia viral antigens.

(D) Vaccination of FVB mice with C+I resulted in a complete rejection of the cancer cells in 7 out of 10 mice by 4 weeks and overall reductions in DB7 tumor size (n = 10 per group; representative images; left). Quantification of the data presented (right).

(B) Representative FACS plot of serum IgG binding of PBS 4-week, iPSC 4-week, C+I 2-week, or C+I 4-week vaccinated mice to embryonic fibroblasts (left panel), iPSCs (middle panel), and DB7 cancer cells (right panel). As a control sample for differentiated cells, a partly differentiated cell culture was included in the iPSC analysis. This is shown by IgG-positive and negative cells, indicating that the IgG binding is specific to the undifferentiated portion of the analyzed cells. C+I 4-week-vaccinated mice showed the best IgG binding to DB7 breast cancer cells.

(A) Optimal vaccination was set to C+I vaccination for 4 weeks, as assessed by percent IgG binding to DB7, without a significant increase in non-specific mouse embryonic feeder (MEF) binding (n = 3 control animals, n = 4 iPSC primed animals, n = 4 C+I primed 2 week, and n = 4 C+I primed 4 week animals, mean ± SEM, ANOVA with Tukey’s multiple comparison test).

We first performed RNA sequencing on 11 different human iPSC clones to compare expression profiles from a selected cancer-related gene list to human ESCs (hESCs), cancer tissues, and healthy tissues ( Figure S1 A). Based on this gene list, we found that human iPSCs cluster with hESCs and the cancer tissues, revealing important gene expression overlap in cancer genes between different cancer types and iPSCs. The upregulation of a subset of these genes was then also validated in murine iPSCs and ESCs ( Figure S1 B). These findings suggest the possibility of using iPSCs in different species to prime the host in developing immunity against known and, perhaps, unknown tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs).

Discussion

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et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. Tumor establishment and progression involve highly proliferative hypoimmunogenic cells that evade the surveillance of the immune system. Therefore, new avenues within the field of cancer treatment are being pursued to target cancer by reactivating the immune system. One way researchers are trying to achieve this is by using chimeric antigen receptors (CARs), with promising results (). The idea behind this therapy is to create a cancer-specific antigen receptor and couple this to an effector cell (e.g., T cell), with newer generations of CARs that might even incorporate the co-stimulatory pathways. However, thus far, results have been mixed, with some patients relapsing, possibly due to loss of expression of the targeted antigen (). One way to circumvent this would be to identify new tumor-specific antigens, but large numbers of tumor antigens are possibly still unknown.

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Nakanishi Y. Vaccination With Irradiated Induced Pluripotent Stem Cells Genetically Engineered To Produce GM-CSF Confers Potent T Cells-Mediated Antitumor Immunity. Pluripotent cells and tissues share known and likely also unknown TSAs and TAAs with cancer cells and therefore could be a potential agent to prime an immune system to target cancer. This modified cell would then function as a surrogate cell type that resembles the targeted cancer type. A few groups have pursued the use of pluripotent cells for priming the immune system in targeting cancer but thus far have not shown efficacy and safety for the treatment of various types of cancer (). In addition, they still rely on the use of ethically concerning ESCs and/or a genetically modified cell line as an adjuvant (), making these treatments less suitable for personalized clinical translation.

Zou, 2005 Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. + effector T cells. The lifespan of these IFN-γ+ effector T cells (8–10 days) would also explain why there was tumor regression after the adoptive transfer of C+I-primed splenocytes in the orthotopic model of breast cancer for the first 2 weeks, after which a small increase in tumor size was seen ( Dooms and Abbas, 2002 Dooms H.

Abbas A.K. Life and death in effector T cells. In this study, we showed that prophylactic immunization of several mouse strains with a non-genetically engineered iPSC-based vaccine produces an effective immune response to multiple cancer types by upregulation of mature APCs in the dLNs with a subsequent increase in helper T cells and cytotoxic T cells locally and, later on, systemically. Interestingly, this led to a systemically favorable T-effector/T-reg ratio, which has been found to reduce tolerizing conditions (). With our adoptive transfer data on transplantation of C+I-primed splenocytes into tumor-bearing mice, we demonstrated the tumor specificity of our iPSC vaccine, which, based on our in vitro data, was likely the result of IFN-γeffector T cells. The lifespan of these IFN-γeffector T cells (8–10 days) would also explain why there was tumor regression after the adoptive transfer of C+I-primed splenocytes in the orthotopic model of breast cancer for the first 2 weeks, after which a small increase in tumor size was seen (). To test whether the immunity created by the vaccine is the result of shared epitopes between iPSCs and cancer cells, we performed adoptive transfer of C+I-primed T cells to breast-cancer-bearing mice and adoptive transfer of TELs to iPSC-inoculated NOD-SCID mice. With these experiments, we were able show that C+I-primed T cells rejected the DB7 breast cancer cells and that the primed TELs were able to reduce teratoma size or stop teratoma formation altogether. This “two-way immunity” demonstrates shared epitopes between iPSCs and cancer cells.

Grupp et al., 2013 Grupp S.A.

Kalos M.

Barrett D.

Aplenc R.

Porter D.L.

Rheingold S.R.

Teachey D.T.

Chew A.

Hauck B.

Wright J.F.

et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. Maude et al., 2014 Maude S.L.

Frey N.

Shaw P.A.

Aplenc R.

Barrett D.M.

Bunin N.J.

Chew A.

Gonzalez V.E.

Zheng Z.

Lacey S.F.

et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. Looking into the early intra-tumor immune response, we found mainly B cells and T cells expressing IL-2, IL-4, and IL-5 with a switch from common T cell clones to rarer vaccine-associated T cell clones. Most of these high-frequency clones vary between the vaccinated mice, suggesting that each mouse mounts a cross-reactive immune response based on different epitopes from the iPSCs. This provides further evidence that iPSCs share a larger repertoire of cancer-related epitopes, indicating that this surrogate cell type could be a potential candidate to limit the chances of immune evasion by the cancer cells as has occasionally been reported in CAR therapy ().

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Rosenberg S.A. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Another issue with CAR therapy is organ toxicity from cytokine storms upon transfusion of CAR T cells (). As we showed in our CBA/J mouse data using the Luminex assay, systemic cytokine levels are low; instead, there is a localized immune response within the tumor similar to the positive control group of tumor rejection. In addition, tissue analysis of our mice at different time points after vaccination did not show any increases in immune cells within heart and kidney tissues compared to negative control groups, nor were elevated levels of anti-nuclear antigen (ANA) IgG seen in serum from C+I-vaccinated mice.

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et al. PD-1 blockade in tumors with mismatch-repair deficiency. +GR1hi myeloid cells, as well as a reduction of tumor-promoting Th17 cells. In this setting, the cancer epitope heterogeneity of iPSCs, combined with the ease of their generation, may make this therapy readily available as adjuvant immunotherapy for multiple cancer types within weeks after diagnosis. As a therapy for established melanomas, the C+I vaccine was not effective in reducing tumor growth, which is likely due to an established immunosuppressive tumor microenvironment that could potentially be remedied by combining the C+I vaccine with checkpoint blockade treatment (). However, as an adjuvant therapy after R1 resection of melanoma, we found that the C+I vaccine reactivated the immune system in rejecting remnant melanoma cells by the systemic upregulation of IL-4-expressing B cells and TNF-α-expressing CD11bGR1myeloid cells, as well as a reduction of tumor-promoting Th17 cells. In this setting, the cancer epitope heterogeneity of iPSCs, combined with the ease of their generation, may make this therapy readily available as adjuvant immunotherapy for multiple cancer types within weeks after diagnosis.

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Fu Y.X. Innate and adaptive immune cells in the tumor microenvironment. This last point is crucial for immunotherapy, because it is commonly known that that the tumor microenvironment could limit effectiveness of tumor immunity by suppressive immune cells residing within the tumor. After debulking of the tumor and disrupting the tumor microenvironment to create an “inflamed” tumor site, immunotherapy should be more effective (). This is demonstrated in our R1 resected melanoma model, which again emphasizes the need for a multi-TSA- and TAA-based vaccine to be readily available at time of tumor resection. Having a surrogate whole-cell vaccine with multiple known (and likely unknown) TSAs and TAAs available at such a short time after diagnosis would allow the priming of the immune system to target large numbers of cancer-specific antigens at a time when cancer cells are most vulnerable.

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Hughes C.C. Of mice and not men: differences between mouse and human immunology. Even though an overlap was seen in murine and human TAA genes, it is important to note the differences in murine and human immunology before extrapolating the above-mentioned data to humans (). Further testing of the C+I vaccine on human samples ex vivo should therefore be performed to show efficacy in humans.

Taken together, our data show the feasibility of creating broad tumor immunity against multiple cancer types using an iPSC-based vaccine that presents the immune system with large quantities of tumor antigens. Compared to current immunotherapy strategies, our iPSC vaccine is capable of reactivating the immune system to target cancers without therapy-associated adverse effects and can be created within a few weeks after diagnosis. These beneficial properties make this iPSC vaccine a potential option for personalized adjuvant immunotherapy shortly after conventional primary treatment of cancer.