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CCR8 mAb treatment mediates potent antitumor activity, suppressing MC38 tumor growth and improving long-term survival.

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CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes–based immunotherapy. Anti-CCR8 mAb therapy synergized with L. monocytogenes–based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies.Significance: Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. Cancer Res; 78(18); 5340–8. ©2018 AACR.

PDF file - 50KB, Protective efficacy maintained by mtrIL-33- and proIL-33-adjuvanted ConE6E7. In a prophylactic study, groups of B6 (10 mice/group) were immunized (three times at three week intervals) with ConE6E7 with or without 7μg of mtrIL-33 or proIL-33 and challenged with 5x104 TC-1 cells one week after last immunization to assess the anti-tumor effects. All vaccination groups prevented tumor growth upon TC-1 implantation.

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PDF file - 58KB, IL-33 expands the frequency of the Ag-specific CD8+ T cells. P14 chimera mice (n=5) were vaccinated twice (on day 0 and day 41) containing GP33 with or without proIL-33. Displayed are representative fluorescent intensity plots of GP33/Ly5.1-specific CD8+ T cells responses in the blood of vaccinated mice at days 14, 21 and 31 after first vaccination and day 48 (day 7 after second immunization). Numbers indicate the percentage of Ag-specific CD8+ T Cells within the total CD8+ T cell populations. Experiments were performed independently at least two times and data are representative of the results.

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Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)–associated model for cancer immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-γ responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4+ and CD8+ T-cell responses, with a large proportion of CD8+ T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8+ T-cell responses and elicit effector–memory CD8+ T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy. Cancer Res; 74(6); 1789–800. ©2014 AACR.

BackgroundRecent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific mutations as a key driver of tumor regression and overall survival. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target a patient’s mutation-derived tumor-specific neoantigens. The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations and control tumor growth by generating neoantigen-specific T cell responses using a pre-clinical mouse tumor model.MethodsWhole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique non-synonymous mutations. The netMHCcons algorithm was used to predict 137 potential neoantigens. We validated 20 immunogenic neoantigens either by peptide immunization followed by ELISPOT or by the presence of CD8+ T cells recognizing the neoantigen peptide following checkpoint inhibitor treatment. Two ADXS-NEO vectors were constructed; Lm20, targeting 20 validated immunogenic neoantigens, and Lm19, targeting most of the non-validated NSMs.ResultsBoth Lm19 & Lm20 significantly slowed tumor growth in C57BL/6 mice compared to control. An accumulation of ADXS-NEO-specific TILs was observed in tumor bearing mice treated with either Lm19 or Lm20. Examination of the tumor microenvironment in Lm19 or Lm20 treated mice revealed a decrease in the frequency and absolute number of Tregs, TAMs, MDSCs, and PD1high exhausted CD8+ T cells as well as an increase in the frequency and absolute number of effector CD8+ T cells, relative to control.ConclusionADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control in mice.

Background There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes–based immunotherapy. Anti-CCR8 mAb therapy synergized with L. monocytogenes–based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies. Significance: Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. Cancer Res; 78(18); 5340–8. ©2018 AACR.

// Daniel O. Villarreal 1 , Diana Chin 1 , Melissa A. Smith 1 , Leopoldo L. Luistro 1 and Linda A. Snyder 1 1 Oncology Discovery, Janssen Research and Development, Spring House, PA 19477, USA Correspondence to: Daniel O. Villarreal, email: dovill33@gmail.com Keywords: vaccines, GITR, PD-1, immunotherapy, immuno-oncology Received: January 05, 2017 Accepted: March 01, 2017 Published: March 27, 2017 ABSTRACT Tumor progression is facilitated immunologically by mechanisms that include low antigen expression, an absence of coimmunostimulatory signals, and the presence of regulatory T cells (Tregs), all of which act to suppress and restrict effector T cells in the tumor. It may be possible to overcome these conditions by a combination of modulatory immunotherapy agents and tumor-antigen targeting to activate and drive effective antitumor T cell responses. Here, we demonstrated that co-administration of aGITR and aPD-1 monoclonal antibodies (mAb) in combination with a peptide vaccine (Vax) in mice bearing established tumors significantly delayed tumor growth and induced complete regression in 50% of the mice. This response was associated with increased expansion and functionality of potent Ag-specific polyfunctional CD8 + T cells, reduced Tregs, and the generation of memory T cells. Tumor regression correlated with the expansion of tumor-infiltrating antigen-specific CD8 + effector memory T cells, as depletion of this cell population significantly reduced the effectiveness of the triple combination Vax/aGITR/aPD-1 therapy. These findings support the concept that dual aGITR/aPD-1 combination with cancer vaccines may be a novel strategy against poorly immunogenic tumors.

Identification of novel molecular adjuvants which can boost and enhance vaccine-mediated immunity and provide dose-sparing potential against complex infectious diseases and for immunotherapy in cancer is likely to play a critical role in the next generation of vaccines. Given the number of challenging targets for which no or only partial vaccine options exist, adjuvants that can address some of these concerns are in high demand. Here, we report that a designed truncated Interleukin-36 gamma (IL-36 gamma) encoded plasmid can act as a potent adjuvant for several DNA-encoded vaccine targets including human immunodeficiency virus (HIV), influenza, and Zika in immunization models. We further show that the truncated IL-36 gamma (opt-36&gamma, t) plasmid provides improved dose sparing as it boosts immunity to a suboptimal dose of a Zika DNA vaccine, resulting in potent protection against a lethal Zika challenge.

Ovarian cancer is frequently diagnosed as peritoneal carcinomatosis. Unlike other tumor locations, the peritoneal cavity is commonly exposed to gut-breaching and ascending genital microorganisms and has a unique immune environment. IL-33 is a local cytokine that can activate innate and adaptive immunity. We studied the effectiveness of local IL-33 delivery in the treatment of cancer that has metastasized to the peritoneal cavity. Direct peritoneal administration of IL-33 delayed the progression of metastatic peritoneal cancer. Prolongation in survival was not associated with a direct effect of IL-33 on tumor cells, but with major changes in the immune microenvironment of the tumor. IL-33 promoted a significant increase in the leukocyte compartment of the tumor immunoenvironment and an allergic cytokine profile. We observed a substantial increase in the number of activated CD4+ T-cells accompanied by peritoneal eosinophil infiltration, B-cell activation and activation of peritoneal macrophages which displayed tumoricidal capacity. Depletion of CD4+ cells, eosinophils or macrophages reduced the anti-tumor effects of IL-33 but none of these alone were sufficient to completely abrogate its positive benefit. In conclusion, local administration of IL-33 generates an allergic tumor environment resulting in a novel approach for treatment of metastatic peritoneal malignancies, such as advanced ovarian cancer.

Mycobacterium tuberculosis infects one third of the world's population. Due to variable efficacy of the Bacille Calmette Guerin (BCG) vaccine, development of novel TB vaccines remains a priority. Here, we demonstrate the protective efficacy of a novel multivalent DNA vaccine, which contains 15 synthetic antigens targeting the Mtb ESX secretion system.

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.

ISG15 is an ubiquitin-like protein induced by type I interferon associated with antiviral activity. ISG15 is also secreted and known to function as an immunomodulatory molecule. However, ISG15's role in influencing the adaptive CD8 T-cell responses has not been studied. Here, we demonstrate the efficacy of ISG15 as a vaccine adjuvant, inducing human papilloma virus (HPV) E7-specific IFNγ responses as well as the percentage of polyfunctional, cytolytic, and effector CD8 T-cell responses. Vaccination with ISG15 conferred remarkable control and/or regression of established HPV-associated tumor-bearing mice. T-cell depletion coupled with adoptive transfer experiments revealed that ISG15 protective efficacy was CD8 T-cell mediated. Importantly, we demonstrate that ISG15 vaccine-induced responses could be generated independent of ISGylation, suggesting that responses were mostly influenced by free ISG15. Our results provide more insight into the immunomodulatory properties of ISG15 and its potential to serve as an effective immune adjuvant in a therapeutic tumor or infectious disease setting.

Identifying new molecular adjuvants that elicit effective vaccine-induced CD8(+) T cell immunity may be critical for the elimination of many challenging diseases including Tuberculosis, HIV and cancer. Here, we report that co-administration of molecular adjuvant IL-33 during vaccination enhanced the magnitude and function of antigen (Ag)-specific CD8(+) T cells against a model Ag, LCMV NP target protein. These enhanced responses were characterized by higher frequencies of Ag-specific, polyfunctional CD8(+) T cells exhibiting cytotoxic characteristics. Importantly, these cells were capable of robust expansion upon Ag-specific restimulation in vivo and conferred remarkable protection against a high dose lethal LCMV challenge. In addition, we demonstrate the ability of IL-33 to amplifying the frequency of Ag-specific KLRG1(+) effector CD8(+) T cells. These data show that IL-33 is a promising immunoadjuvant at improving T cell immunity in a vaccine setting and suggest further development and understanding of this molecular adjuvant for strategies against many obstinate infectious diseases and cancer.

Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

// Daniel O. Villarreal 1 , Michael J. Allegrezza 1 , Melissa A. Smith 1 , Diana Chin 1 , Leopoldo L. Luistro 1 and Linda A. Snyder 1 1 Oncology Discovery, Janssen RD vaccines; GITR; immunotherapy; CD8 T cells Received: June 30, 2017 Accepted: November 05, 2017 Published: November 24, 2017 ABSTRACT Mounting evidence demonstrates that CD8 + CD122 + T cells have suppressive properties with the capacity to inhibit T cell responses. Therefore, these cells are rational targets for cancer immunotherapy. Here, we demonstrate that CD122 monoclonal antibody (mAb; aCD122) therapy significantly suppressed tumor growth and improved long-term survival in tumor-bearing mice. This therapeutic effect correlated with enhanced polyfunctional, cytolytic intratumoral CD8 + T cells and a decrease in granulocytic myeloid-derived suppressor cells (G-MDSCs). In addition, aCD122 treatment synergized with a vaccine to augment vaccine-induced antigen (Ag)-specific CD8 + T cell responses, reject established tumors and generate memory T cells. Furthermore, aCD122 mAb synergized with an anti-GITR (aGITR) mAb to confer significant control of tumor growth. These results suggest CD122 might be a promising target for cancer immunotherapy, either as a single agent or in combination with other forms of immunotherapy.

Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

Interleukin 33 (IL-33) has emerged as a cytokine that can exhibit pleiotropic properties. Here we examine IL-33 for its immunoadjuvant effects in an HPV-associated cancer immune therapy model in which cell-mediated immunity is critical for protection. It is known that two biologically active forms of IL-33 exist: full-length IL-33 and mature IL-33. The potential ability of both isoforms to act as vaccine adjuvants to influence the CD4 Th1 and CD8 T cell immune responses has not been well defined. We show that both isoforms of IL-33 are capable of enhancing potent antigen (Ag)-specific effector and memory T cell immunity in vivo in a DNA vaccine setting. We also show that while both forms of IL-33 drove robust IFN-γ responses, neither form drove high secretion of IL-4 or any elevation of IgE levels. Moreover, both isoforms augmented vaccine-induced Ag-specific polyfunctional CD4+ and CD8+ T cell responses, with a large proportion of CD8+ T cells undergoing cytolytic plurifunctional degranulation. Therapeutic studies indicated that established TC-1-bearing mice undergo rapid and complete regression after therapeutic vaccination with both IL-33 adjuvant isoforms used in conjunction with an HPV DNA vaccine. Furthermore, using the P14 transgenic mouse model, we show that IL-33 can significantly expand the magnitude of Ag-specific CD8+ T cell responses and elicit bonafide effector-memory CD8+ T cells. Overall, the data suggests the potential use of these two IL-33 isoforms as immunoadjuvant candidates in future vaccination against other pathogens and in the context of anti-tumor immune-based therapy.

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

The human body has developed an elaborate defense system against microbial pathogens and foreign antigens. However, particular microbes have evolved sophisticated mechanisms to evade immune surveillance, allowing persistence within the human host. In an effort to combat such infections, intensive research has focused on the development of effective prophylactic and therapeutic countermeasures to suppress or clear persistent viral infections. To date, popular therapeutic strategies have included the use of live-attenuated microbes, viral vectors and dendritic-cell vaccines aiming to help suppress or clear infection. In recent years, improved DNA vaccines have now re-emerged as a promising candidate for therapeutic intervention due to the development of advanced optimization and delivery technologies. For instance, genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as codelivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of potent heterologous prime-boost regimens has also provided significant contributions to DNA vaccine immunogenicity. Herein, the authors will focus on these recent improvements to this synthetic platform in relation to their application in combating persistent virus infection.

Multivalent DNA vaccines that are delivered by electroporation (EP) through muscle tissue provide a novel method for eliciting immunity against tuberculosis (TB) as well as a broad range of diseases including HIV and cancers. Proper plasmid construction containing suitable protective TB antigens capable of evoking desired vaccine-induced responses would lead to the appropriate induction of both humoral and cellular immunity. DNA vaccines are safe and of low cost in comparison to traditional vaccines while also providing potentially effective prophylactic or therapeutic modalities against currently untreatable diseases. Here, we describe the steps for developing a rational multivalent TB DNA vaccine delivered with intramuscular EP in mice.

Multivalent DNA vaccines that are delivered by electroporation (EP) through muscle tissue provide a novel method for eliciting immunity against tuberculosis (TB) as well as a broad range of diseases including HIV and cancers. Proper plasmid construction containing suitable protective TB antigens capable of evoking desired vaccine-induced responses would lead to the appropriate induction of both humoral and cellular immunity. DNA vaccines are safe and of low cost in comparison to traditional vaccines while also providing potentially effective prophylactic or therapeutic modalities against currently untreatable diseases. Here, we describe the steps for developing a rational multivalent TB DNA vaccine delivered with intramuscular EP in mice.

Introduction: Virtually all tumors contain somatic mutations that can result in novel antigenic sequences that may be targeted by the host cellular immune response. Some of these mutations occur in preferential regions of specific genes commonly referred to as hotspot mutations. Hotspot mutations are commonly shared by cancer patients both within and across multiple tumor types. These hotspot mutations often confer loss or gain of function contributing to oncogenesis, which makes them promising therapeutic targets. One such mutation commonly found in several human tumor types is an aspartic acid substitution for glycine at position 12 (G12D) in KRAS. This same mutation occurs in the CT26 murine colorectal tumor model. To determine if expression of the KRAS G12D sequence in a bacterial immunotherapy vector can control tumor growth in the CT26 murine model, the Advaxis Listeria monocytogenes (Lm)-based platform was engineered to express a 21-amino acid KRAS sequence peptide containing the G12D mutation (Lm-Hot KRAS_G12D). In addition, we evaluated control of tumor growth using an ADXS-HOT construct (ADXS-503) that expresses multiple shared human hotspot and tumor-associated antigens, including the G12D KRAS. The ADXS-HOT clinical program is comprised of several Lm-based immunotherapies designed to target multiple shared hotspot and tumor-associated antigens commonly found in specific cancer types. In this study, we demonstrate control of tumor growth in a mouse model by targeting a commonly shared hotspot mutation using an Lm-based immunotherapy. Results: We show that the Lm-HOT KRAS_G12D therapy significantly delayed tumor growth and improved long-term survival in the murine CT26 colon carcinoma model. This response was associated with an increase in the frequency of tumor infiltrating antigen-specific CD8 T cells and γδ T cells within the tumor microenvironment and a decrease in the frequency of intratumoral regulatory T cells (Tregs). Furthermore, tumor-specific CD8 T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Interestingly, our proprietary ADXS-503 (a clinical ADXS-HOT construct) which includes KRAS G12D as one of its multiple targets, was also capable of significantly suppressing tumor growth in the CT26 tumor model. Conclusion: These results suggest that our ADXS-HOT platform is a promising approach to target shared hotspot mutations. That ADXS Lm constructs targeting a single hotspot mutation can significantly control tumor growth whether it is in a single or multi-target construct. These data describe an exciting translatable discovery with the potential for broad utility across multiple tumor types and patients who share common hotspot mutations. Citation Format: Daniel Villarreal, Brandon Coder, Susan Armington, Andrew L'Huillier, Cristina Mottershead, Elena Filippova, Nithya Thambi, Kim Ramos, David Balli, Robert Petit, Michael Princiotta. Targeting shared hotspot cancer mutations with a Listeria monocytogenes immunotherapy induce potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-149.

Introduction: Recent advances in the field of cancer immunotherapy have identified CD8+ T cell responses against tumor-specific neoantigens as a key driver of tumor regression and prolonged survival. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to generate immune responses against mutation-derived tumor-specific neoantigens. Advaxis' Lm-based immunotherapies consist of live highly-attenuated bacterial vectors that are bioengineered to secrete a fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific neoantigens that harbor nonsynonymous point mutations (NSMs). The objective of this study is to demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific point mutations to generate neoantigen-specific T cells and control tumor growth. Results: Whole-exome sequencing of the MC38 mouse tumor cell line identified 2870 unique NSMs. Among these, the IC50 of 138 NSMs were predicted to be less than 500 nM by the netMHCcons algorithm. We evaluated the immunogenicity of 37 NSMs, and found that 12 immunogenic NSMs elicited a CD8+ T cell response following peptide immunization. Moreover, we identified 10 additional immunogenic NSMs in MC38-bearing mice treated with a check point inhibitor. Altogether, we identified 22 immunogenic and 23 non-immunogenic NSMs. Two ADXS-NEO vectors were constructed, Lm-19 & Lm-20, targeting 19 non-immunogenic and 20 immunogenic NSMs respectively. The ability of Lm-19 and Lm-20 to control MC38 tumor growth was evaluated in C57BL/6J mice. We found that both Lm-19 & Lm-20 led to an accumulation of neoantigen-specific CD8+ TILs and significantly slowed tumor growth. Moreover, both Lm-19 and Lm-20 decreased the frequency and absolute number of intratumoral Tregs, TAMs, and MDSCs and increased the frequency and absolute number of effector CD8+ T cells. Interestingly, expression of PD-L1 was decreased in TAMs and MDSCs and the frequency and total number of granzyme A+ CD8+ effector T cells was increased. Furthermore, the proportion of phenotypically exhausted PD-1hiLAG3+ TILs was decreased. Together, these data suggest the tumor microenvironment in mice receiving Lm-19 and Lm-20 becomes more cytotoxic and less suppressive. Conclusion: ADXS-NEO is a potent immunotherapy capable of driving immune responses against tumor-specific mutations and leading to tumor control. The effectiveness of the Lm platform is demonstrated by the generation of neoantigen-specific T cells to peptide sequences that were identified as “non-immunogenic” using a conventional peptide-adjuvant immunization. This study is a clear demonstration that T cell mediated anti-tumor responses can be generated by targeting tumor-derived NSMs with the ADXS-NEO Listeria monocytogenes vector. Citation Format: Brandon Coder, Hyewon Phee, Cristina Mottershead, Dipti Kelkar, Elena Filippova, Xiaoming Ju, Bryan Vander Lugt, Olga Pryshchep, Justin Lesch, Xian Liu, Jason DeVoss, Keegan Cooke, Claret Liu, Jinghui Zhan, Petia Mitchell, Kim Ramos, Daniel O. Villarreal, Jim Johnston, Robert Petit, Michael Princiotta. Neoantigens that fail to elicit measurable T cell responses following peptide immunization can control tumor growth when delivered using a Listeria-based immunotherapy platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-150.

Introduction: Neoantigens derived from tumor-specific mutations have been shown to drive tumor specific CD8+ T cell responses leading to tumor regression and extending overall survival. Frameshift mutations are estimated to generate up to nine times more neoantigens per mutation compared to in-frame mutations. However, it is not clear if vaccination against frameshift mutations induces neoantigen-specific CD8+ T cell responses that result in control of tumor growth. ADXS-NEO is a personalized Listeria monocytogenes (Lm)-based immunotherapy designed to target mutation-derived tumor-specific neoantigens. Advaxis' Lm-based immunotherapies consist of live attenuated bacterial vectors that are bioengineered to secrete an antigen-adjuvant fusion protein consisting of a truncated non-hemolytic fragment of listeriolysin O, which has adjuvant properties, and tumor-specific antigens. Here, we demonstrate the feasibility of using the ADXS-NEO platform to target tumor-specific frameshift mutations in order to generate neoantigen-specific T cells that control tumor growth. Results: Whole-exome sequencing of the CT26 and MC38 mouse tumor cell lines identified 30 and 31 unique frameshift mutations respectively. Individual frameshift mutations ranged in size from 12 to as many as 150 amino acids (aa). Lm vectors targeting the two longest frameshift mutations were constructed for each tumor model. The therapeutic efficacy of Lm vectors expressing either a single 57 aa (Lm-57) or a single 150 aa (Lm-150) MC38 frameshift mutation were evaluated in C57BL/6J mice. Both Lm vectors generated multiple unique frameshift-specific TILs and slowed tumor growth. Furthermore, we evaluated the tumor microenvironment following Lm-57 or Lm-150 treatment and observed a decrease in the frequency and absolute number of Tregs, TAMs, and MDSCs and an increase in the frequency and absolute number of total cytotoxic granzyme A+ effector CD8+ T cells. Similarly, Lm vectors expressing either a 64 aa (Lm-64) or a 93 aa (Lm-93) CT26 frameshift mutation were evaluated in the CT26 tumor model. Both Lm-64 and Lm-93 significantly controlled tumor growth. Additionally, an influx of neoantigen-specific TILs and a significant decrease in the frequency of intratumoral Tregs was observed. Conclusion: ADXS-NEO induced potent immune responses against tumor-specific frameshift mutations and controlled tumor growth. Advaxis' Lm platform is able to target frameshift mutations ≥150 aa and generate multiple neoantigen-specific T cells per frameshift. ADXS-NEO controls tumor growth via multiple mechanisms, including the generation of tumor-specific cytotoxic TILs, by secreting tumor-derived neoantigens directly into dendritic cells and by attenuating the suppressive tumor microenvironment. Citation Format: Brandon Coder, Daniel O. Villarreal, Susan Armington, Elena Filippova, Andrew L'Huillier, Dipti Kelkar, Xiaoming Ju, Cristina Mottershead, David Balli, Kim Ramos, Hyewon Phee, Jim Johnston, Robert Petit, Michael Princiotta. Targeting frameshift mutations with a Listeria monocytogenes immunotherapy drives neoantigen-specific antitumor immunity in the MC38 and CT26 mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-148.

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone linear closed DNA [(dbDNA)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4(+) and CD8(+) T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

Tuberculosis (TB) still remains a major public health issue despite the current available vaccine for TB, Bacille Calmette Guerin (BCG). An effective vaccine against TB remains a top priority in the fight against this pandemic bacterial infection. Adequate protection against TB is associated with the development of TH1-type and CD8(+) T cell responses. One alarmin cytokine, interleukin 33 (IL-33), has now been implicated in the development of both CD4(+) TH1 and CD8(+) T cell immunity. In this study, we determined whether the administration of IL-33 as an adjuvant, encoded in a DNA plasmid, could enhance the immunogenicity of a TB DNA vaccine. We report that the co-immunization of IL-33 with a DNA vaccine expressing the Mycobacterium Tuberculosis (Mtb) antigen 85B (Ag85B) induced robust Ag85B-specific IFNγ responses by ELISpot compared to Ag85B alone. Furthermore, these enhanced responses were characterized by higher frequencies of Ag85B-specific, multifunctional CD4(+) and CD8(+) T cells. Vaccination with IL-33 also increased the ability of the Ag85B-specific CD8(+) T cells to undergo degranulation and to secrete IFNγ and TNFα cytokines. These finding highlights IL-33 as a promising adjuvant to significantly improve the immunogenicity of TB DNA vaccines and support further study of this effective vaccine strategy against TB.

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous β-herpes virus that infects 60–95 % of adults worldwide. Infection is a major cause of congenital abnormalities in newborns, contributes to development of childhood cerebral palsy and medulloblastoma, can result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. While CMV has been increasingly associated with numerous inflammatory diseases and cancers, only recently has it been correlated with increased risk of heart disease in adults, the number-one killer in the USA. These data, among others, suggest that subclinical CMV infection, or microinfection, in healthy individuals may play more of a causative role than an epiphenomenon in development of CMV-associated pathologies. Due to the myriad of diseases and complications associated with CMV, an efficacious vaccine would be highly valuable in reducing human morbidity and mortality as well as saving billions of dollars in annual health-care costs and disability adjusted life years (DALY) in the developing world. Therefore, the development of a safe efficacious CMV vaccine or immune therapy is paramount to the public health. This review aims to provide a brief overview on aspects of CMV infection and disease and focuses on current vaccine strategies. The use of new synthetic DNA vaccines might offer one such approach to this difficult problem.

For many years IL-33 has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Interestingly, IL-33 has now emerged as a cytokine with a plethora of pleiotropic properties. Depending on the immune cells targeted by IL-33, it is reported to not only promote Th2 immunity, but also to induce T helper type 1 (Th1) immunity. Furthermore, recent studies have revealed that IL-33 can activate CD8(+) T cells. These new studies provide evidence for its beneficial role in antiviral and antitumor immunity. Here we review the evidence of IL-33 to drive protective T cell immunity plus its potential use as an adjuvant in vaccination and tumor therapy.

Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses’ ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8 T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases.

Ebola is a hemorrhagic fever virus that is responsible for the severe, ongoing outbreak in West Africa, with over 21000 confirmed cases and a mortality rate of >39%. Control of the outbreak has been a considerable challenge as there are no approved vaccines or therapies. Recent reports suggest that the outbreak is more severe than previously thought and investigating alternative immunization approaches would be of great benefit. We generated 3 DNA vector vaccine candidates expressing the Zaire ebolavirus (EBOV) glycoprotein (GP): 2 vaccines designed based on consensus sequences of EBOV GPs (1976-2014) and a 3rd matched construct to a 2014 Guinea strain. Immunogenicity and protection was first evaluated in mice, followed by studies in non-human primates (NHP). BALB/c mice received a single immunization of each DNA vaccine (40mg), delivered by intramuscular injection followed by electroporation (IM-EP). We also co-administered 2 or 3 construct formulations of DNA vaccines as an alternative strategy to broaden efficacy against antigenically distant EBOV strains. All 3 single and all co-formulated vaccines generated strong T cell responses, including GP-specific polyfunctional CD4+ and CD8+ T cells secreting IFNγ, TNFα, and IL2. BALB/c mice (n=10/group) were challenged with a lethal dose of a heterologous mouse-adapted Mayinga 1976 EBOV strain. The two consensus DNA vaccines and 2 and 3 vaccine combinations were 100% protective in mice. The matched vaccine afforded 90% protection. Total GP-specific IgG levels were high in all surviving mice and low in unprotected animals suggesting that titers may correlate with therapeutic efficacy. As all 3 DNA vaccines were immunogenic and no antigen interference was observed, we next investigated immunogenicity of the 3 DNA vaccines in rhesus macaques following a DNA-DNA prime-boost immunization regimen. Macaques (n=5/group) received by IM-EP delivery:1) matched vaccine (4mg), 2) 3-construct formulation administered at a single injection site (2mg/vaccine), 3) 3-construct formulation delivered at separate injection sites (2mg/vaccine), or 4) 3-construct formulation combined with IL12 adjuvant (2mg/vaccine). Each immunization was followed by 3 subsequent DNA boosts at 1 month intervals. All groups had high GP-specific total IgG titers post-1st boost immunization that continued to increase over subsequent boosts and detectable T-cell responses. Overall, these studies demonstrate that a DNA-DNA prime-boost regimen can effectively induce seroconversion in NHPs and further studies will serve to evaluate protective immunogenicity. This approach not only serves as a tool to better understand the immune responses associated with protection against EBOV infection, but also has potential for translation towards human applications.

Human papillomavirus (HPV) infection is associated with 99% of cervical cancers and approximately 20% of head and neck squamous cell carcinomas. HPV E6- and E7-specific T cell immunity has been considered a critical characteristic of an effective therapeutic vaccine designed to control and eliminate established pre-existing HPV infections and associated cancers. The use of molecular adjuvants is considered an important tool to increase the potency of DNA vaccines further. IL-12 has shown to drive cellular immune responses by aiding the priming and expansion of CD8 T cells. We have previously demonstrated that a highly optimized engineered DNA vaccine targeting HPV 16 and 18 E6/E7 (pGX3001 and pGX3002) elicited potent TH1 and cytotoxic cellular immune responses in a Phase I clinical trial. Here, we sought to determine whether the HPV vaccine-induced antitumor immunity could be further enhanced by the addition of IL12 as an adjuvant.We first demonstrated that IL-12 could enhance HPV16 E6 and E7-specific cellular responses measured by ELISpot in mice (average of 1037 vs 504 SFU/106 splenocytes). Then we performed an in vivo tumor therapy study to determine whether IL-12 could enhance the therapeutic efficacy of pGX3001 in TC-1 tumor bearing C57BL/6 mice. The results induced that mice immunized with pGX3001+IL-12 exhibited smaller tumors compared to those in the no adjuvant group. Thirty days post tumor transplantation, 9 out of 10 mice in the pGX3100+IL-12 group showed complete tumor regression, while there were only 4 out of 10 mice in the pGX3001 group that showed complete tumor regression, indicating that the addition of plasmid-based mouse IL-12 enhanced the vaccine-induced antitumor immunity in a murine model. Furthermore, an additional study in a more relevant, non-human primate model was performed to ensure that the vaccine-induced cellular immune responses could also be augmented by using a rhesus macaque IL-12 plasmid. The data indicated that the addition of IL-12 increased the magnitude of vaccine-induced cellular immune responses about two fold post third immunization (average of 2711 SFU vs 1400 SFU/106 PBMCs as measured by ELISpot). The HPV antigen-specific memory responses in the HPV+IL-12 group were increased more than four-fold compared to the responses in the unadjuvanted group (average of 2570 vs 577 SFU/106 PBMCs). Flow cytometric analysis revealed the induction of both HPV-specific CD4 and CD8 T cells that efficiently increased IFN-γ responses. Both adjuvant and unadjuvanted groups showed an increase in effector and central memory CD8 T cells after immunization, while the IL-12 group exhibited stronger effector memory CD8 T cell responses. Taken together, these data support the further development of this HPV DNA vaccine in combination with IL12 as a cancer immunotherapy candidate.

The use of immune plasmid adjuvants encoding cytokine, chemokine or immune modulators to tailor the vaccine induced response is a strength of DNA vaccine approaches. This has been established through the recent HVTN080 trial which demonstrated the potency of pIL-12 in increasing cellular responses above that of DNA only vaccination. Due to its role in both innate and adaptive immunity, the co-delivery of plasmid encoded CD40 ligand (CD40L) could increase DNA vaccine responses. In its natural form, CD40L can occur as either a surface bound form or a cleaved/solubilized form. Thus, we sought to determine if different forms of pCD40L can influence cellular and humoral responses when co-delivered with a HPV16 DNA vaccine expressing the oncogenic proteins E6 and E7. Mice were immunized with HPV DNA with or without synthetic optimized plasmids expressing various forms of CD40L. Mice which received the soluble form of CD40L (sCD40L) exhibited significantly higher antigen specific CD8+ T cell responses including IFN-γ, IL-2 and TNF-α expression. These responses were maintained into memory. On the other hand, the surface bound as well as the wild type form blunted the vaccine induced responses compared to vaccine alone. Time course analysis revealed that 11 days after primary immunization, CD8+ tetramer specific (H-2Db HPV16 E7 (RAHYNIVTF)) T cells in mice immunized with sCD40L averaged around 18% compared to vaccine alone at 4%. These responses were partially dependent on CD4+ T cell help. Upon therapeutic tumor challenge, mice immunized with sCD40L displayed significant tumor regression compared to vaccine alone or naive animals. We have also seen similar vaccine induced immune responses when sCD40L was combined with an HIV Envelope expressing DNA vaccine as well as an H1N1 flu DNA vaccine. These results demonstrate the power of using an immune plasmid adjuvant encoding a synthetic optimized sCD40L in a DNA vaccine. Additional studies in other models are important for this approach to be considered for possible clinical development.

First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

Recent studies are beginning to show that IL-33 cytokine activities exceed does of the Th2 immunity, and can promote Th1 immune responses. However, the potential ability of IL-33 to act as vaccine adjuvants to influence the CD4 Th1 and CD8 T cell immune responses has not been well defined. We have currently examined IL-33 for its immunoadjuvant effects in an HPV-associated cancer immune therapy murine model in which cell-mediated immunity is critical for protection. We showed that IL-33 was capable of enhancing potent antigen (Ag)-specific effector and memory T cell immunity in vivo in a DNA vaccine setting. Additionally, IL-33 augmented vaccine-induced Ag-specific polyfunctional CD4+ sand CD8+ T cell responses, with a large proportion of CD8+ T cells undergoing cytolytic plurifunctional degranulation. Therapeutic studies indicated that established TC-1-bearing mice underwent rapid and complete regression after therapeutic vaccination with IL-33 in conjunction with an HPV DNA vaccine. Furthermore, we show that IL-33 can significantly expand the magnitude of Ag-specific CD8+ T cell responses and elicit effector-memory CD8+ T cells. Given these significant findings we also explored IL-33 adjuvant effects in conjunction with HIV and LCMV DNA vaccine. Interestingly, we demonstrate that IL-33 also increases the antigen-specific T cell immune responses when co-delivered with HIV and that IL-33 modulates LCMV-Tetramer-specific memory CD8 T cell differentiation that provided protection against a lethal challenge of LCMV. Overall, these results lay the groundwork of IL-33 as an immunoadjuvant candidate in future vaccination in the context of both antitumor and antiviral immunotherapy.

Adjuvants are critical components of most clinically used vaccines and act as substances to enhance adaptive immune responses to an antigen. Here we examine a new immunoadjuvant, interferon stimulated gene 15 (ISG15), for its adjuvant effects in an HPV-associated cancer immune therapy model where cell-mediated immunity is crucial for protection. ISG15 is an ubiquitin-like protein induced by type I interferon and associated with antiviral activity. Recent studies of ISG15 have re-established that ISG15 is also a secreted molecule with cytokine-like properties. However, the potential ability of ISG15 to influence the Th1 and CD8 T cell immune responses and act a vaccine adjuvant, has not been well defined. To this end, we developed a highly optimized DNA-vector encoding the ISG15 protein and evaluated its immunogenicity to increase the cellular immune responses in combination with a DNA consensus-based fusion HPV16 E6/E7 construct. We demonstrate that ISG15 can enhance Ag-specific Th1 memory responses to HPV16 E6/E7 antigen by IFN-γ ELISpot analysis. Moreover, ISG15 elicited strong HPV16 Ag-specific polyfunctional CD8+ T cells that secrete both anti-viral IFN-γ and/or TNF-α cytokines. Vaccination conferred remarkable 100% protection against challenge with HPV E6 and E7-expressing tumors. Overall, these findings lay the groundwork of ISG15 as a new immunoadjuvant candidate in the context of cancer immunotherapy. Citation Format: Daniel Villarreal, Megan C. Wise, Jian Yang, David B. Weiner. Immunoadjuvant ISG15 enhances human papillomavirus 16 antigen-specific cell-mediated antitumor immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2014-LB-257

3098 Background: Antigen specific CD4+ and CD8+ T cell responses have been generated in 78-90% of volunteers in two recent HPV and HIV clinical studies using our highly optimized DNA immunogens del...