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T-cell recognition of cancer neoantigens is important for effective immune-checkpoint blockade therapy, and an increasing interest exists in developing personalized tumor neoantigen vaccines. Previous studies utilizing RNA and long-peptide neoantigen vaccines in preclinical and early-phase clinical studies have shown immune responses predominantly driven by MHC class II CD4+ T cells. Here, we report on a preclinical study utilizing a DNA vaccine platform to target tumor neoantigens. We showed that optimized strings of tumor neoantigens, when delivered by potent electroporation-mediated DNA delivery, were immunogenic and generated predominantly MHC class I–restricted, CD8+ T-cell responses. High MHC class I affinity was associated specifically with immunogenic CD8+ T-cell epitopes. These DNA neoantigen vaccines induced a therapeutic antitumor response in vivo, and neoantigen-specific T cells expanded from immunized mice directly killed tumor cells ex vivo. These data illustrate a unique advantage of this DNA platform to drive CD8+ T-cell immunity for neoantigen immunotherapy.

Expression of ESR1 by PCR and lack of responsiveness of tumor cells used in vivo.

TC1, LLC and ID8 neoantigen list

Supplementary Figure 1: Expression of human and mouse consensus MAGE-A plasmids in vitro. Supplementary Figure 2: Consensus mouse MAGE-A vaccine induces low CD4+ T cells in C57Bl/6 mice. Supplementary Figure 3: Consensus mouse MAGE-A vaccine induces immune responses to one dominant epitope. Supplementary Figure 4: MAGE-A vaccine induced memory T cell responses. Supplementary Figure 5: Evaluation of organ toxicity after MAGE-A immunization. Supplementary Figure 6: Expression of mouse MAGE-A in a panel of mouse tumor cell lines and in autochthonous melanomas. Supplementary Figure 7: Immune responses in spleen and tumor of autochthonous tumor bearing mice. Supplementary Figure 8: Impact of MAGE-A vaccine in YUMM1.7 subcutaneous tumor model.

Supplementary Figure 1 shows the anti-mouse CTLA-4 DMAb design. Supplementary Figure 2 shows the efficacy of the recombinant 9D9 antibody in the Sa1N tumor model. Supplementary Figure 3 shows that the mouse anti-mouse CTLA-4 DMAb induces immune memory and protection from tumor re-challenge. Supplementary Figure 4 shows the efficacy of the mouse anti-mouse CTLA-4 DMAb delivered at an earlier time point. Supplementary Figure 5 shows that the anti-mouse CTLA-4 DMAb induces T cell infiltration into tumors. Supplementary Figure 6 shows the efficiency of CD4 and CD8 depletion antibodies.

Supplementary Figure 1: Construct design and expression. Supplementary Figure 2: Comparison of Native and SynCon FAP vaccines in C57Bl/6 mice. Supplementary Figure 3: Comparison of Native and SynCon FAP vaccines in Balb/c mice. Supplementary Figure 4: Efficacy of FAP vaccine in therapeutic prostate and breast tumor models. Supplementary Figure 5: Alteration of the tumor microenvironment using hyaluronidase is less effective at synergizing with DNA vaccines. Supplementary Figure 6: Immune responses from mice receiving combination mTERT + FAP vaccination. Supplementary Figure 7: Impact of SynCon FAP vaccine on immune cell subsets by flow cytometry. Supplementary Figure 8: FAP expression and immune cell infiltration in patients.

Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti–CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.Significance: DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies. Cancer Res; 78(22); 6363–70. ©2018 AACR.

Supplemental Methods

Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines.Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data.Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8+ and CD4+ immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen–specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8+ T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8+ T-cell infiltration.Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190–201. ©2018 AACR.

Supplemental Figure 1. (A) Cytotoxicity of FSHCER or mock transduced T-cells of OVCAR-3 luciferase tumor cells measured by luciferase detection after co-culture of 18 hours. (B) Gating strategy followed for the flow cytometry based cytotoxicity assay. After gating on the singlet cells we could gate the tumor cells and exclude T-cells by FSC and SSC (confirmed by the no Tcell control) and measure the percentage of living cells as double negatives from Annexin-V and 7AAD. Supplemental Figure 2. (A) immunohistochemistry showing a section CAOV3 tumor stained with FSHR18 antibody followed by secondary anti-mouse (FSHR18) or secondary alone (Negative). 20x, scale bar: 1 um. (B) Western blot and qPCR showing FSHR expression of different ovarian cancer and breast cancer tumor cell lines. (C) Tumor volume of three ovarian patient-derived xenograft tumors grown in the flank of NOD-SCID mice (n=2 mice per tumor, one case-one control, single experiment) injected intratumorally with 10 million FSHCER or mock transduced T-cells (arrows mark time of T-cell injection). Common axis used for easier cross comparison. Supplemental Figure 3. (A) FSHR mRNA of FACS-sorted ID8-Defb29/Vegf-a/Fshr from the peritoneal cavity of orthotopic ID8-Defb29/Vegf-a/Fshr-bearing mice treated with either FSHCER, mock transduced T-cells or PBS. (B) Normalized real-time quantitative-PCR of FSHR expression in the human healthy ovarian tissue shown in Figure 3A, a human serous ovarian carcinoma specimen and 2 additional cell lines. (C) Absence of expression of the FSHR in untransduced ID8-Defb29/Vegf-a cells, compared to FSHR-transduced clones (40 PCR cycles).

Purpose: To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells.Experimental Design: FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells.Results: FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo. In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1–dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor–transduced T cells away from targeted tumor cells.Conclusions: T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441–53. ©2016 AACR.

Purpose:Cancer/testis antigens have emerged as attractive targets for cancer immunotherapy. Clinical studies have targeted MAGE-A3, a prototype antigen that is a member of the MAGE-A family of antigens, in melanoma and lung carcinoma. However, these studies have not yet had a significant impact due to poor CD8+ T-cell immunogenicity, platform toxicity, or perhaps limited target antigen availability. In this study, we develop an improved MAGE-A immunogen with cross-reactivity to multiple family members.Experimental Design:In this study, we analyzed MAGE-A expression in The Cancer Genome Atlas and observed that many patients express multiple MAGE-A isoforms, not limited to MAGE-A3, simultaneously in diverse tumors. On the basis of this, we designed an optimized consensus MAGE-A DNA vaccine capable of cross-reacting with many MAGE-A isoforms, and tested immunogenicity and antitumor activity of this vaccine in a relevant autochthonous melanoma model.Results:Immunization of this MAGE-A vaccine by electroporation in C57Bl/6 mice generated robust IFNγ and TNFα CD8+ T-cell responses as well as cytotoxic CD107a/IFNγ/T-bet triple-positive responses against multiple isoforms. Furthermore, this MAGE-A DNA immunogen generated a cross-reactive immune response in 14 of 15 genetically diverse, outbred mice. We tested the antitumor activity of this MAGE-A DNA vaccine in Tyr::CreER;BRAFCa/+;Ptenlox/lox transgenic mice that develop melanoma upon tamoxifen induction. The MAGE-A DNA therapeutic vaccine significantly slowed tumor growth and doubled median mouse survival.Conclusions:These results support the clinical use of consensus MAGE-A immunogens with the capacity to target multiple MAGE-A family members to prevent tumor immune escape.

Neoantigens are tumor-specific antigens that arise due to somatic mutations in the DNA of tumor cells. They represent ideal targets for cancer immunotherapy since there is minimal risk for on-target, off-tumor toxicities. Additionally, these are foreign antigens that should be immunogenic due to lack of central immune tolerance. Tumor neoantigens are predominantly passenger mutations, which do not contribute to tumorigenesis. In cases of multi-focal or metastatic tumors, different foci can have significantly different mutation profiles. This suggests that it is important to target as many neoantigens as possible to better control tumors and target multi-focal tumors within the same patient. Herein, we report a study targeting up to 40 neoantigens using a single DNA plasmid. We observed significant plasticity in the epitope strings arranged in the vaccine with regard to immune induction and tumor control. Different vaccines elicited T cell responses against multiple epitopes on the vaccine string and controlled growth of multi-focal, heterogeneous tumors in a therapeutic tumor challenge. Additionally, the multi-epitope antigens induced long-term immunity and rejected a tumor re-challenge several weeks after the final vaccination. These data provide evidence that DNA-encoded long antigen strings can be an important tool for immunotherapeutic vaccination against neoantigens with implications for other in vivo-delivered antigen strings., Graphical abstract, Weiner and colleagues report on a synthetic DNA vaccine targeting 40 neoantigens. The immunogenicity of individual epitopes was unaffected by their positioning on long vaccine strings or the size of vaccine constructs. These vaccines generated robust, long-term T cell immunity and controlled growth of heterogeneous multi-focal tumors.

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αβ and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αβ T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αβ T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αβ and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common “a determinant region” of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directing in vivo antibody expression. In vivo delivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cells in vitro. Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.

BackgroundTumor neoantigens are epitopes derived from tumor-specific mutations. Such mutations can be incorporated in personalized vaccines to prime T cell responses against tumor specific antigens. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth.MethodsTwo resection samples from a patient with IDH+ MGMT-methylated anaplastic astrocytoma were subject to whole exome and transcriptome sequencing. Epitopes derived from 27 neoantigens and 3 shared tumor-associated antigens were prioritized and included in a personalized vaccine. The patient was treated with surgery, radiotherapy and temozolomide starting June 2018 and received the first dose of the personalized vaccine in June 2019 under a compassionate use single patient IND application with the FDA.ResultsAs of July 23rd, 2021, the patient has received 11 doses of the DNA personalized vaccine. No serious adverse events have been reported. Related adverse events are limited to grade 1 injection site reactions. The patient remains progression-free 37 months after surgery and 25 months after starting vaccination. Three weeks following the 3rd dose, a hyperintense image on the tumor bed was identified, which disappeared on the following MRI, 2 weeks following dose 5, being catalogued as pseudo progression. Ex vivo ELISpot have identified T cell responses to 28/30 epitopes (93.3%), including 25/27 (92.6%) neoantigens and 3/3 (100%) shared antigens. Flow cytometry analysis has determined that T cell responses are 92.3% CD8 and 69.2% CD4 (30.8% CD8 only; 61.5% both CD8 and CD4; and 7.7% CD4 only).ConclusionsThis compassionate use treatment in an adjuvant setting demonstrates manufacturing feasibility, safety, tolerability, immunogenicity, and suggests potential for persistent clinical response of DNA encoded personalized vaccines. The data supports further investigation of DNA-encoded personalized vaccines into newly diagnosed high-grade gliomas.Ethics ApprovalThe study was approved by Washington University's IRB. The participant gave informed consent before taking part in the study.

BackgroundHepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death. Immune checkpoint inhibitors targeting PD-1 have limited activity in HCC as monotherapy, with response rates ranging from 14–17%. Tumor neoantigens derived from tumor-specific mutations can be incorporated into personalized therapeutic cancer vaccines to generate tumor-specific T cell immunity, potentially priming the immune system for anti-PD1 therapy. DNA vaccines have been shown to elicit strong CD8 and CD4 T cell responses in preclinical and clinical trials. GNOS-PV02 is a personalized DNA vaccine, encoding up to 40 patient-specific neoantigens. In the GT-30 trial, it is used in combination with INO-9012 (plasmid-encoded IL-12) and pembrolizumab for the treatment of advanced HCC.MethodsGT-30 is a single-arm phase I/II clinical trial to assess the safety, immunogenicity, and preliminary efficacy of GNOS-PV02 in combination with INO-9012 and pembrolizumab in patients with advanced HCC. Twenty-four patients are anticipated to be enrolled. Patients are recruited upon diagnosis or during first-line treatment with tyrosine kinase inhibitors (TKI). Tumors are biopsied for exome and transcriptome sequencing, and peripheral blood collected for germline sequencing and histogenetics. The tumor specific vaccine is designed, optimized and manufactured during first-line therapy. Each vaccine encodes up to 40 neoantigens. After progression or intolerance with first-line therapy, patients commence concurrent personalized vaccine and pembrolizumab. GNOS-PV02 + INO-9012 are administered Q3w for the first 4 doses and Q9w thereafter. Pembrolizumab is delivered Q3w.ResultsWe performed a data cut-off on the first 12 patients. The median age was 66 years (range 55–75 years). GNOS-PV02 + INO-9012 with pembrolizumab has had no reported DLTs or drug related SAEs. The most common treatment-related AE were grade 1 fatigue (25%) and grade 1 injection site reactions (17%). By including up to 40 epitopes in the vaccine we were able to target all neoantigens present in 83% of the patients. The objective response rate was 25% (3/12 partial response, 5/12 stable disease, 4/12 progressive disease). Analysis of the TCR repertoire in peripheral blood and tumor tissue identified novel and significantly expanded T cell clones post-vaccination in all patients analyzed. Many of the novel peripheral T cell clones were also identified to have trafficked to the TME at week 9, potentially mediating the observed tumor regressions.ConclusionsThese data demonstrate the potential of GNOS-PV02 + INO-9012 with pembrolizumab to target multiple neoepitopes, and provide initial support for the safety and efficacy of this regimen in patients with advanced HCC.Trial RegistrationNCT04251117Ethics ApprovalThe study obtained IRB approval (IRB) and all patients signed informed consent prior to taking part in the clinical trial. NZCR EC: 20/NTA/6; JHU: IRB00227771; Mount Sinai: HS#: 20–00076

Research question The study aimed to determine whether IVF or intrauterine growth restriction (IUGR) result in short neonatal telomeres, which could explain the higher risk of cardiovascular and metabolic disease described in these populations. Design This was an observational, analytical, cross-sectional, prospective study with controls in a tertiary hospital. The main outcome was to determine the leukocyte telomere length in 126 newborns and their mothers (n = 109). Newborns were conceived spontaneously or by IVF, and uncomplicated and IUGR pregnancies were studied. Telomere lengths were measured using high-throughput telomere quantitative fluorescent in-situ hybridization. Results There was no difference in average telomere length between newborns conceived by IVF or those with IUGR and spontaneously conceived healthy newborns (P = 0.466 and P = 0.732, respectively); this remained after controlling for confounders (P = 0.218 and P = 0.991, respectively). Mothers of newborns with IUGR had a shorter average telomere length than women with uncomplicated pregnancies (P = 0.023), which was confirmed after controlling for age, body mass index and smoking habit (P = 0.034). Conclusions The results support the safety of IVF and IUGR in terms of the postnatal health of the newborns. The shorter telomeres of IUGR mothers may represent a higher cardiovascular risk, which would have clinical implications under the stress of pregnancy in otherwise healthy adults.

T-cell recognition of cancer neoantigens is important for effective immune-checkpoint blockade therapy, and an increasing interest exists in developing personalized tumor neoantigen vaccines. Previous studies utilizing RNA and long-peptide neoantigen vaccines in preclinical and early-phase clinical studies have shown immune responses predominantly driven by MHC class II CD4+ T cells. Here, we report on a preclinical study utilizing a DNA vaccine platform to target tumor neoantigens. We showed that optimized strings of tumor neoantigens, when delivered by potent electroporation-mediated DNA delivery, were immunogenic and generated predominantly MHC class I–restricted, CD8+ T-cell responses. High MHC class I affinity was associated specifically with immunogenic CD8+ T-cell epitopes. These DNA neoantigen vaccines induced a therapeutic antitumor response in vivo, and neoantigen-specific T cells expanded from immunized mice directly killed tumor cells ex vivo. These data illustrate a unique advantage of this DNA platform to drive CD8+ T-cell immunity for neoantigen immunotherapy.

Checkpoint inhibitors (CPI) have revolutionized the treatment of many solid tumors. However, difficulties in production, stability, the requirement of frequent high doses for antibody administration and long intravenous administration are recurring issues. Synthetically designed DNA-encoded monoclonal antibodies (DMAbs) are a novel delivery method for antibody therapy which could potentially address many of these issues, simplifying design and implementation of MAb-based therapies. DMAbs delivered through plasmid DNA injection and electroporation have been used in preclinical models for the treatment or prophylaxis of infectious diseases, cancer and cardiovascular disease. Our group has recently reported that immune checkpoint blockers can be optimized and delivered in vivo advancing further DMAb technology by optimization, expression and in vivo functional characterization of anti-CTLA4 antibodies. Here we report optimization, expression and binding of DMAbs based on anti-PD1 CPI and discuss the potential of DMAbs in checkpoint immunotherapy.

Ovarian cancer (OC) represents the deadliest gynecologic malignancy. Despite important advances in the field of OC therapy, recurrent OC still has very poor prognosis with a median survival of 1 year. Due to the close interaction between the ovarian cancer cells and tumor microenvironment, development of treatment strategies which not only target the tumor cells but also the components of the tumor microenvironment hold significance. Notably, a prime obstacle in the development of therapies is to identify targets with specific expression limited to the tumor surface and not the healthy tissues. The follicle-stimulating hormone receptor (FSHR) is one such target with selective expression in ovarian granulosa cells and thus, a potentially important therapeutic target in OC. Therefore, we developed monoclonal antibodies against FSHR and focused on studies of the most potent of these reagents. Anti-FSHR antibody bound to diverse FSHR expressing ovarian serous and clear cell adenocarcinoma cells suggesting these antibodies could be used to specifically target OC. We then designed a novel DNA encoded bispecific T cell engager targeting FSHR (FSHRxCD3) and evaluated this bispecific in therapeutic models for the treatment of OC. FSHRxCD3 bispecific in the presence of human PBMCs was highly specific in killing FSHR positive ovarian tumor lines. However, T cell approaches alone may not be fully effective in treating OC, referred as Immunologically “Cold” Tumors. Hence, we hypothesized that engaging the other components of immune system, would provide better tumor control. Increasing lines of evidence suggest that OC is receptive to Natural killer (NK) cell attack. We designed antibodies against human Siglec-7, an inhibitory receptor present on human NK cells and showed they could bind to NK cells. We then used these to create a novel class of bispecific NK engager (NKE) that simultaneously targets both Siglec-7 and FSHR (Siglec-7xFSHR). This NKE was potent at killing FSHR positive OC targets in both in vitro and in vivo assays. Multiple Ovarian tumors including BRCA mutated and PARPi resistant ovarian cancer cells could be targeted by this immune therapy. Our data demonstrate the therapeutic potential of these two novel bispecific molecules and initial studies suggest that their combination may be valuable in patients with OC. Citation Format: Devivasha Bordoloi, Pratik Bhojnagarwala, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Alfredo Perales-Puchalt, Ryan P. O’Connell, Xizhou Zhu, Elizabeth M. Parzych, Rugang Zhang, Mohamed Abdel-Mohsen, David B. Weiner. Immunotherapy of ovarian cancer targeting FSHR by innate and adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4262.

Epstein Barr Virus (EBV) latent infection is associated with multiple types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV+ malignancies, suggesting additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV-associated gastric carcinoma (EBVaGC) and EBV+ nasopharyngeal carcinoma (NPC) and has not been evaluated as an antigen in the context of therapeutic immunization. Here, we developed a synthetic DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and polyfunctional effector T cell responses in vivo. We observed significant antigen-specific increases of IFN-γ+, IFN-γ+ TNF-α+, and IFN-γ+ TNF-α+ IL-2+ populations in CD8+ T cells from the pBARF1 immunized animals in both C57BL/6 and BALB/c mice. Novel BARF1 expressing carcinoma lines that grow in immune-competent animals were developed as surrogates for human EBV+ tumors. We confirmed that BARF1 expression in these new cancer cell lines, MC38-BARF1 and CT26-BARF1, was within the range of human EBV+ cancer cell lines, SNU719 and C666-1. Immunization with pBARF1 plasmid demonstrated impact of tumor progression through induction of CD8+ T cell in these BARF1+ carcinoma models. We also observed long-term antitumor immunity, at least up to 446 days, against BARF1 in a CT26-BARF1 rechallenge study. Using IVIS in vivo imaging, we observed that pBARF1 immunized animals rapidly controlled and cleared cancer cells. These studies demonstrate the potential for pBARF1 immunity to induce antigen-specific immune responses impacting tumor progression. Further study of this immune target and these EBV+ tumor models is likely important as part of therapeutic approaches for EBV+ malignancies. Citation Format: Xizhou Zhu, Alfredo Perales-Puchalt, Krzysztof Wojtak, Ziyang Xu, Kun Yun, Pratik S. Bhojnagarwala, Devivasha Bordoloi, Daniel H. Park, Kevin Liaw, Mamadou A. Bah, Paul M. Lieberman, Ebony N. Gary, Ami Patel, David B. Weiner. DNA immunotherapy targeting BARF1 demonstrates therapeutic impact in novel murine carcinoma models expressing EBV latent antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3573.

2638 Background: Tumor neoantigens are epitopes derived from tumor-specific mutations that can be incorporated in personalized vaccines to prime T cell responses. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth. Methods: Paired blood and tumor biopsy samples were collected from a patient with hepatocellular carcinoma before and after treatment with GNOS-PV02 (DNA neoantigen targeted vaccine) + plasmid IL-12 + pembrolizumab. Treatment resulted in a partial response with a decrease in tumor size of 44% by RECIST (168 mm to 94 mm). TCRbeta sequencing was performed on all 4 samples and single cell TCR and transcriptome sequencing was performed from T cells isolated from the post-treatment blood sample. Newly identified TCRs in blood and tumor after vaccination were inserted into an expression vector and used to generate engineered TCR T cells. Engineered TCR T cells were tested against the neoantigens included in the vaccine and their responses characterized by flow cytometry. Results: We identified 67,893 new clones in PBMC after vaccination, 3 of which comprised between 0.1 to 1% of the total T cell clones. Moreover, we identified 5126 new clones in the tumor post vaccination, out of these, 3878 (75.68%) were not found within the patient’s pre vaccination PBMCs and 556 (10.86%) were identified within the pre vaccination PBMC pool. Importantly, of the newly identified T cells infiltrating the tumor post vaccination, we observed high frequency TCR clones of which 44 and 7 clones were above 0.1% and 1%, respectively. The majority of the newly identified T cell clones were CD8 T cells (68.75%) with an activated phenotype. Importantly, the 6 most expanded clones in blood were identified to be activated CD8+CD69+ T cells (81.82%). Engineered TCR T cells generated encoding the TCRs of these newly identified CD8 T cells showed activation when exposed to the tumor neoantigens encoded in the neoantigen DNA vaccine GNOS-PV02. Conclusions: GNOS-PV02, a neoantigen DNA vaccine, in combination with plasmid IL-12 and pembrolizumab resulted in expansion of newly identified T cells, primarily activated CD8, which trafficked to the tumor. These new tumor infiltrating T cells showed TCR specificity against tumor neoantigens encoded in GNOS-PV02 and may account for the observed objective decrease in tumor size.

Purpose: Cancer/testis antigens have emerged as attractive targets for cancer immunotherapy. Clinical studies have targeted MAGE-A3, a prototype antigen that is a member of the MAGE-A family of antigens, in melanoma and lung carcinoma. However, these studies have not yet had a significant impact due to poor CD8+ T-cell immunogenicity, platform toxicity, or perhaps limited target antigen availability. In this study, we develop an improved MAGE-A immunogen with cross-reactivity to multiple family members. Experimental Design: In this study, we analyzed MAGE-A expression in The Cancer Genome Atlas and observed that many patients express multiple MAGE-A isoforms, not limited to MAGE-A3, simultaneously in diverse tumors. On the basis of this, we designed an optimized consensus MAGE-A DNA vaccine capable of cross-reacting with many MAGE-A isoforms, and tested immunogenicity and antitumor activity of this vaccine in a relevant autochthonous melanoma model. Results: Immunization of this MAGE-A vaccine by electroporation in C57Bl/6 mice generated robust IFNγ and TNFα CD8+ T-cell responses as well as cytotoxic CD107a/IFNγ/T-bet triple-positive responses against multiple isoforms. Furthermore, this MAGE-A DNA immunogen generated a cross-reactive immune response in 14 of 15 genetically diverse, outbred mice. We tested the antitumor activity of this MAGE-A DNA vaccine in Tyr::CreER;BRAFCa/+;Ptenlox/lox transgenic mice that develop melanoma upon tamoxifen induction. The MAGE-A DNA therapeutic vaccine significantly slowed tumor growth and doubled median mouse survival. Conclusions: These results support the clinical use of consensus MAGE-A immunogens with the capacity to target multiple MAGE-A family members to prevent tumor immune escape.

Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti–CTLA-4 monoclonal antibodies in vivo. With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 μg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 μg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay ex vivo. In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.Significance: DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies. Cancer Res; 78(22); 6363–70. ©2018 AACR.

Prostate cancer is a prevalent cancer in men and consists of both indolent and aggressive phenotypes. While active surveillance is recommended for the former, current treatments for the latter include surgery, radiation, chemo and hormonal therapy. It has been observed that the recurrence in the treated patients is high and results in castration resistant prostate cancer for which treatment options are limited. This scenario has prompted us to consider immunotherapy with synthetic DNA vaccines, as this approach can generate antigen-specific tumor-killing immune cells. Given the multifocal and heterogeneous nature of prostate cancer, we hypothesized that synthetic DNA vaccines targeting different prostate specific antigens are likely to induce broader and improved immunity who are at high risk as well as advanced clinical stage of prostate cancer, compared to a single antigen approach. Utilizing a bioinformatics approach, synthetic enhanced DNA vaccine (SEV) constructs were generated against STEAP1, PAP, PARM1, PSCA, PCTA and PSP94. Synthetic enhanced vaccines for prostate cancer antigens were shown to elicit antigen-specific immune responses in mice and the anti-tumor activity was evident in a prostate tumor challenge mouse model. These studies support further evaluation of the DNA tools for immunotherapy of prostate cancer and perhaps other cancers.

Arginase is a complex and unique enzyme that plays diverse roles in health and disease. By metabolizing arginine, it can shape the outcome of innate and adaptive immune responses. The immunomodulatory capabilities of arginase could potentially be applied for local immunosuppression or induction of immune tolerance. With the use of an enhanced DNA delivery approach, we designed and studied a DNA-encoded secretable arginase enzyme as a tool for immune modulation and evaluated its immunomodulatory function in vivo. Strong immunosuppression of cluster of differentiation 4 (CD4) and CD8 T cells, as well as macrophages and dendritic cells, was observed in vitro in the presence of an arginase-rich supernatant. To further evaluate the efficacy of DNA-encoded arginase on in vivo immunosuppression against an antigen, a cancer antigen vaccine model was used in the presence or absence of DNA-encoded arginase. Significant in vivo immunosuppression was observed in the presence of DNA-encoded arginase. The efficacy of this DNA-encoded arginase delivery was examined in a local, imiquimod-induced, psoriasis-like, skin-inflammation model. Pretreatment of animals with the synthetic DNA-encoded arginase led to significant decreases in skin acanthosis, proinflammatory cytokines, and costimulatory molecules in extracted macrophages and dendritic cells. These results draw attention to the potential of direct in vivo-delivered arginase to function as an immunomodulatory agent for treatment of local inflammation or autoimmune diseases., Graphical Abstract, Arginase is a unique enzyme that plays a critical role in regulating the outcome of innate and adaptive immune responses. The immunomodulatory capabilities of arginase enzyme could potentially be applied for local inflammation or autoimmune diseases. Here, we report on a DNA-encoded secretable arginase enzyme as a tool for therapeutic immunomodulation.

In article number https://doi.org/10.1002/advs.201902802, David B. Weiner, Daniel W. Kulp, and co‐workers demonstrate that synthetic DNA delivery and adaptive electroporation can launch de novo assembly of different nanoparticle vaccines in the hosts to elicit significantly improved responses while reducing required doses. Next‐generation designer vaccines can now be rapidly evaluated in vivo to facilitate clinical translation for the betterment of human and animal health.

Prostate cancer (PCa) represents the most predominant cancer type among males with an estimated 191,930 cases diagnosed in 2020 with 33,330 deaths globally. Notably, different treatment modalities such as surgery, chemo, radiation and hormone therapy exhibited improved outcomes in early stage PCa patients; however, there exist high recurrence rate in the treated patients and leads to castration resistant prostate cancer, the treatment choices for which are highly restricted. This scenario has pressed us to consider synthetic DNA vaccine-based immunotherapy approach, with ability to induce antigen-specific tumor-killing immune cells. Considering the high heterogeneity of prostate cancer cells, we hypothesized that synthetic DNA vaccines targeting multiple prostate specific antigens would elicit improved immune responses in those who are at high risk as well as advanced stages of prostate cancer. Therefore, we generated synthetic enhanced DNA immunogens as DNA constructs (SEV) targeting STEAP1, PAP, PARM1, PSCA, PCTA and PSP94 through utilization of bioinformatics approaches. The SEVs for prostate cancer antigens elicited potent cellular immunity as well as polyfunctionality of antigen-specific T cells in mice. Further, they were capable of generating robust humoral immune responses to each antigen. The anti-tumor activities of the SEVs were studied in prostate tumor which showed that the induced immune responses delayed tumor progression and enhanced infiltration of anti-tumor CD8+ T cells in the tumor microenvironment in a mouse model challenge system. These data highlight further study of these novel immunogens as synthetic DNA immunogens for immunotherapy of PCa, and perhaps other human cancers. Citation Format: Devivasha Bordoloi, Peng Xiao, Hyeree Choi, Michelle Ho, Alfredo Perales-Puchalt, Makan Khoshnejad, J.Joseph Kim, Laurent Humeau, Alagarsamy Srinivasan, David B. Weiner, Kar Muthumani. Novel synthetic DNA vaccines in prostate cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1915.

TPS2680 Background: Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death. Immune checkpoint inhibitors targeting PD-1 have limited activity in HCC as monotherapy, with response rates ranging from 14-17%. Tumor neoantigens derived from tumor-specific mutations can be incorporated into personalized therapeutic cancer vaccines to prime T cell responses, potentially enhancing responses to anti-PD1 therapy. DNA vaccines have been shown to elicit strong CD8 and CD4 T cell responses in preclinical and clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth. GNOS-PV02 is a personalized DNA vaccine, encoding up to 40 patient-specific neoantigens. In the GT-30 trial, it is used in combination with INO-9012 (plasmid-encoded IL-12) and pembrolizumab for the treatment of advanced HCC. Methods: The GT-30 trial (NCT04251117) is a single-arm phase I/II clinical trial to assess the safety, immunogenicity, and preliminary efficacy of GNOS-PV02 in combination with INO-9012 and pembrolizumab in patients with advanced HCC. Twenty-four patients are anticipated to be enrolled. Patients are recruited upon diagnosis or during first-line treatment with tyrosine kinase inhibitors (TKI). Tumors are biopsied for exome and transcriptome sequencing. The tumor specific vaccine is designed, optimized and manufactured during first-line therapy. Each vaccine encodes up to 40 neoantigens, which includes all detected neoantigens for the majority of HCC patients. After progression or intolerance with first-line therapy, patients can commence trial therapy with concurrent personalized vaccine and pembrolizumab. GNOS-PV02 + INO-9012 are administered Q3w for the first 4 doses and Q9w thereafter until disease progression. Pembrolizumab is delivered Q3w until disease progression. Immunogenicity of each of the vaccine epitopes will be determined by ex vivo ELISpot and flow cytometry. Clinical activity is assessed by RECIST1.1 at baseline and every 9 weeks. Serial biopsies will be obtained at 9 weeks and upon disease progression to evaluate changes in the exome, transcriptome and changes to the tumor microenvironment. Clinical trial information: NCT04251117.

e14561 Background: Tumor neoantigens are epitopes derived from tumor-specific mutations that may be incorporated in personalized vaccines to prime T cell responses. DNA vaccines delivered with electroporation have recently shown strong CD8 and CD4 T cell responses in clinical trials. In preclinical studies, DNA-encoded neoantigen vaccines have shown induction of CD8 T cells against 50% of predicted high affinity epitopes with the ability to impact tumor growth. Methods: Two resection samples from a patient with IDH+ MGMT-methylated anaplastic astrocytoma were subject to whole exome and transcriptome sequencing. Epitopes derived from 27 neoantigens and 3 shared tumor-associated antigens were prioritized and included in a personalized vaccine. The patient was treated with surgery, radiotherapy and temozolomide starting June 2018 and received the first dose of the personalized vaccine in June 2019 under a compassionate use single patient IND application with the FDA. Results: As of February 10, 2021, the patient has received 10 doses of the DNA personalized vaccine. No serious adverse events have been reported. Related adverse events are limited to grade 1 injection site reactions. The patient remains progression-free 32 months after surgery and 20 months after starting vaccination. Three weeks following the third dose, a hyperintense image on the tumor bed was identified, which disappeared on the following MRI, 2 weeks following dose 5, being catalogued as pseudo progression. Ex vivo ELISpot have identified T cell responses to 21/30 epitopes (70%), including 18/27 (67%) neoantigens and 3/3 (100%) shared antigens. Flow cytometry analysis has determined that T cell responses are 92.3% CD8 and 69.2% CD4 (30.8% CD8 only; 61.5% both CD8 and CD4; and 7.7% CD4 only). Conclusions: This compassionate use treatment in an adjuvant setting demonstrates manufacturing feasibility, safety, tolerability, immunogenicity, and suggests potential for persistent clinical response of DNA encoded personalized vaccines. The data supports further investigation of DNA-encoded personalized vaccines into newly diagnosed high-grade gliomas.

Prostate-specific membrane antigen (PSMA) is expressed at high levels on malignant prostate cells and is likely an important therapeutic target for the treatment of prostate carcinoma. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines as well as passive administration of PSMA-specific monoclonal antibodies (mAb). Conventional mAb immunotherapy has numerous logistical and practical limitations, including high production costs and a requirement for frequent dosing due to short mAb serum half-life. In this report, we describe a novel strategy of antibody-based immunotherapy against prostate carcinoma that utilizes synthetic DNA plasmids that encode a therapeutic human mAb that target PSMA. Electroporation-enhanced intramuscular injection of the DNA-encoded mAb (DMAb) plasmid into mice led to the production of functional and durable levels of the anti-PSMA antibody. The anti-PSMA produced in vivo controlled tumor growth and prolonged survival in a mouse model. This is likely mediated by antibody-dependent cellular cytotoxicity (ADCC) effect with the aid of NK cells. Further study of this novel approach for treatment of human prostate disease and other malignant conditions is warranted.

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.