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Supplementary Table S1

PDF file - 81K, Characterization of tumor infiltrating T cells following treatment with systemic DNA vaccination and locally applied imiquimod.

Purpose:Choriocarcinoma is most commonly gestational (androgenetic or biparental) but can be of germ cell origin or can develop as a component of a somatic neoplasm (genetically related to the patient). The latter type are aggressive neoplasms for which the underlying genetic alterations are not well characterized.Experimental Design:To investigate the relationship between the different components of somatic neoplasms with choriocarcinomatous elements, the genetic differences between gestational and nongestational tumors, and identify potential targetable alterations, we analyzed 23 samples from 11 tumors, including five gynecologic-type somatic neoplasms with choriocarcinomatous differentiation (two to three different components each) and six pure choriocarcinomas, for somatic mutations, single-nucleotide polymorphisms, and PD-L1 expression.Results:In mixed tumors, gynecologic-type carcinoma components demonstrated lineage-characteristic and lineage-specific alterations, with choriocarcinomatous components sharing some of these as well as demonstrating novel alterations, supporting a clonal relationship with divergent differentiation of the choriocarcinoma from the somatic carcinoma. TP53 mutation only occurred in nongestational tumors. Diffuse PD-L1 expression was characteristic of choriocarcinoma in both pure and mixed tumors but not seen in the gynecologic-type carcinoma components.Conclusions:Given that the somatic carcinomatous and choriocarcinomatous components of mixed tumors have distinct genetic alterations and biomarker expression, separate analysis of these components is required to guide targeted therapy. High PD-L1 expression suggests a role for checkpoint inhibitor–based immunotherapy in tumors with a choriocarcinoma component. The underlying mechanisms by which cancer stem cells reprogram and initiate trophoblastic retrodifferentiation in some somatic tumors warrant further investigation.

Supplementary Figure S5

Supplemental Figure Legend

Supplementary Tables S2A and S2B

Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7.Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance.Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract.Conclusions: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. Clin Cancer Res; 20(21); 5456–67. ©2014 AACR.

PDF file - 70K, E7-specific T cells efficiently migrated from circulation to the genital tract following imiquimod application.

Supplementary Table and Figure legends

Supplementary Figure 3 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Supplementary Figure 2 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Ovarian cancer is responsible for the highest mortality rate among patients with gynecologic malignancies. Therefore, there is an emerging need for innovative therapies for the control of advanced ovarian cancer. Immunotherapy has emerged as a potentially plausible approach for the control of ovarian cancer. In the current study, we have generated heat shock protein 70 (Hsp70)-secreting murine ovarian cancer cells that express luciferase (MOSEC/luc). Hsp70 has been shown to target and concentrate antigenic peptides in dendritic cells and is also able to activate dendritic cells. We characterized the antigen-specific immune response and the antitumor effect of the MOSEC/luc cells expressing Hsp70 using noninvasive luminescence images to measure the amount of ovarian tumors in the peritoneal cavity of mice. We found that mice challenged with MOSEC/luc cells expressing Hsp70 generate significant antigen-specific CD8+ T-cell immune responses. Furthermore, we also found that mice vaccinated with irradiated MOSEC/luc cells expressing Hsp70 generate significant therapeutic effect against MOSEC/luc cells. In addition, we have shown that CD8+, natural killer, and CD4+ cells are important for protective antitumor effect generated by irradiated tumor cell–based vaccines expressing Hsp70. Moreover, we also found that CD40 receptor is most important, followed by Toll-like receptor 4 receptor, for inhibiting in vivo tumor growth of the viable MOSEC/luc expressing Hsp70. Thus, the use of Hsp70-secreting ovarian tumor cells represents a potentially effective therapy for the control of lethal ovarian cancer. [Cancer Res 2007;67(20):10047–57]

Supplementary Figure 1 from Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion

Supplementary Figure 1 from Cancer Immunotherapy Using Irradiated Tumor Cells Secreting Heat Shock Protein 70

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8+ (CD8+) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8+ T cells through its interaction with the α4β1 integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with α4β1 integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC. [Cancer Res 2007;67(4):1832–41]

Supplementary Figure 2 from Cancer Immunotherapy Using Irradiated Tumor Cells Secreting Heat Shock Protein 70

Background The spread of SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19), has been characterized as a worldwide pandemic. Currently, there are few preclinical animal models that suitably represent infection, as the main point of entry to human cells is via human angiotensin-converting enzyme 2 (ACE2) which is not present in typical preclinical mouse strains. Additionally, SARS-CoV-2 is highly virulent and unsafe for use in many research facilities. Here we describe the development of a preclinical animal model using intranasal administration of ACE2 followed by non-infectious SARS-CoV-2 pseudovirus (PsV) challenge. Methods To specifically generate our SARS-CoV-2 PsV, we used a lentivirus system. Following co-transfection with a packaging plasmid containing HIV Gag and Pol, luciferase-expressing lentiviruses, and a plasmid carrying the SARS-CoV-2 spike protein, SARS-CoV-2 PsVs can be isolated and purified. To better understand and maximize the infectivity of SARS-CoV-2 PsV, we generated PsV carrying spike protein variants known to have varying human ACE2 binding properties, including 19 deletion (19del) and 19del + D614G. Results Our system demonstrated the ability of PsVs to infect the respiratory passage of mice following intranasal hACE2 transduction. Additionally, we demonstrate in vitro and in vivo manipulability of our system using recombinant receptor-binding domain protein to prevent PsV infection. Conclusions Our PsV system is able to model SARS-CoV-2 infections in a preclinical mouse model and can be used to test interventions or preventative treatments. We believe that this method can be extended to work in various mouse strains or to model infection with different coronaviruses. A simple in vivo system such as our model is crucial for rapidly and effectively responding to the current COVID-19 pandemic in addition to preparing for future potential coronavirus outbreaks.

BackgroundFor centuries, microbial-based agents have been investigated as a therapeutic modality for the treatment of cancer. In theory, these methods would be cheap to produce, broadly applicable in a wide array of cancer types, and could synergize with other cancer treatment strategies. We aimed to assess the efficacy of combining microbial-based therapy usingSalmonellaSL7207 with interleukin-2 (IL-2), a potent immunostimulatory agent, in the treatment of murine colon carcinoma.MethodsFemale BALB/c mice were implanted subcutaneously with CT26 tumors, a model of colon carcinoma. Mice bearing tumors were selected and administered Albumin-IL-2 (Alb-IL2), a fusion protein, for further analysis of anticancer effect.ResultsWe demonstrated thatSalmonellaSL7207, a genetically modified strain ofSalmonella entericaserovar Typhimurium, preferentially accumulates in the tumor microenvironment, potentiating it to stimulate localized innate immunity. We delivered IL-2 as a fusion protein, Alb-IL2, which we demonstrate to have preferential accumulation properties, bringing it to the tumor and secondary lymphoid organs. Treatment of tumor-bearing mice withSalmonella + Alb-IL2 leads to superior tumor control and enhanced overall survival compared to controls. When assessing immunological factors contributing to our observed tumor control, significantly enhanced T cell population with superior effector function was observed in mice treated withSalmonella + Alb-IL2. We confirmed that these T cells were indispensable to the observed tumor control through antibody-mediated T cell depletion experiments.ConclusionsThese findings highlight the ability ofSalmonella + Alb-IL2 to serve as a novel therapeutic approach to induce T cell-mediated antitumor immunity and exert long-term tumor control in a murine model of cancer.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads, variants with enhanced virulence and transmissibility have emerged. Although in vitro systems allow rapid characterization, they do not fully recapitulate the dynamic interaction of virions and neutralizing antibodies in the airway. Here, we demonstrate that the N501Y variant permits respiratory infection in unmodified mice. We utilize N501Y to survey in vivo pseudovirus infection dynamics and susceptibility to reinfection with the L452R (Los Angeles), K417N + E484K (South Africa), and L452R + K417N + E484Q (India) variants. Human coronavirus disease 2019 (COVID-19)+ or vaccinated antibody isotypes, titers, variant receptor binding domain (RBD) binding, and neutralization potential are studied, revealing numerous significant correlations. Immune escape of the K417N + E484K variant is observed because infection can be appreciated in the nasopharynx, but not lungs, of mice transferred with low-antibody-tier plasma. Conversely, near-complete protection is observed in animals receiving high-antibody-tier plasma, a phenomenon that can only be appreciated in vivo., Graphical abstract, Lam et al. develop a model of SARS-CoV-2 infection in laboratory mice. This allows researchers to study the threat of emerging variants in a more physiological context than by using cell culture systems. Interactions between SARS-CoV-2 variants and immunity are explored in the airway of mice.

The phospholipid phosphatidylserine (PS) is naturally maintained on the cytoplasmic side of the plasma membrane. Independent of apoptosis, PS is redistributed to the surface of CD8 T cells in response to TCR-mediated activation. Annexin V (AnnV) is a protein known to bind PS with high affinity and has been effectively utilized to anchor antigen to the surface of CD8 T cells. To expand these studies, we aimed to exploit TCR activation driven PS exposure as a target to deliver cytokine, namely interleukin-2 (IL-2), to the surface of CD8 T cells. This was accomplished using a novel chimeric fusion protein of annexin V and interleukin 2 (AnnV-IL2). In vitro analysis revealed that AnnV-IL2 is able to specifically bind PS on the T cell surface following TCR stimulation. Consequently, AnnV-IL2 proved to be significantly more effective at enhancing T cell activation compared to recombinant IL-2. In vivo, AnnV-IL2 promotes robust expansion of antigen-specific cells capable of interferon gamma (IFNγ) production when administered following peptide vaccination. Importantly, upon antigen rechallenge, AnnV-IL2 treatment mice demonstrated a stronger secondary expansion, indicating durability of AnnV-IL2 mediated responses. Our data supports the use of AnnV-IL2 to modulate antigen-specific T cell immunity and demonstrates that the PS-AnnV axis is a feasible mechanism to target diverse cargo to CD8 T cells.

Purpose: Choriocarcinoma is most commonly gestational (androgenetic or biparental) but can be of germ cell origin or can develop as a component of a somatic neoplasm (genetically related to the patient). The latter type are aggressive neoplasms for which the underlying genetic alterations are not well characterized. Experimental Design: To investigate the relationship between the different components of somatic neoplasms with choriocarcinomatous elements, the genetic differences between gestational and nongestational tumors, and identify potential targetable alterations, we analyzed 23 samples from 11 tumors, including five gynecologic-type somatic neoplasms with choriocarcinomatous differentiation (two to three different components each) and six pure choriocarcinomas, for somatic mutations, single-nucleotide polymorphisms, and PD-L1 expression. Results: In mixed tumors, gynecologic-type carcinoma components demonstrated lineage-characteristic and lineage-specific alterations, with choriocarcinomatous components sharing some of these as well as demonstrating novel alterations, supporting a clonal relationship with divergent differentiation of the choriocarcinoma from the somatic carcinoma. TP53 mutation only occurred in nongestational tumors. Diffuse PD-L1 expression was characteristic of choriocarcinoma in both pure and mixed tumors but not seen in the gynecologic-type carcinoma components. Conclusions: Given that the somatic carcinomatous and choriocarcinomatous components of mixed tumors have distinct genetic alterations and biomarker expression, separate analysis of these components is required to guide targeted therapy. High PD-L1 expression suggests a role for checkpoint inhibitor–based immunotherapy in tumors with a choriocarcinoma component. The underlying mechanisms by which cancer stem cells reprogram and initiate trophoblastic retrodifferentiation in some somatic tumors warrant further investigation.

A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor (albGM-CSF) molecule that harnesses albumin’s long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1β release from Mtb-infected dendritic cells and macrophages, higher IL-1β levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.

Using a comprehensive next-generation sequencing pipeline (143 genes), Oncomine Comprehensive v.2, we analyzed genetic alterations on a set of vulvar squamous cell carcinomas (SCCs) with emphasis on the primary and metastatic samples from the same patient, to identify amenable therapeutic targets. Clinicopathologic features were reported and genomic DNA was extracted from 42 paraffin-embedded tumor tissues of 32 cases. PD-L1 expression was evaluated in 20 tumor tissues (10 cases with paired primary and metastatic tumors). Fifteen (88%) of 17 successfully analyzed HPV-unrelated SCCs harbored TP53 mutations. 2 different TP53 mutations had been detected in the same tumor in 4 of 15 cases. Other recurrent genetic alterations in this group of tumors included CDKN2a mutations (41%), HRAS mutations (12%), NOTCH1 mutations (12%) and BIRC3 (11q22.1-22.2) amplification (12%). Six HPV-related tumors harbored PIK3CA, BAP1, PTEN, KDR, CTNNB1, and BRCA2 mutations, of which, one case also contained TP53 mutation. Six cases showed identical mutations in paired primary site and distant metastatic location and four cases displayed different mutational profiles. PD-L1 expression was seen in 6 of 10 primary tumors and all 6 paired cases showed discordant PD-L1 expression in the primary and metastatic sites. Our results further confirmed the genetic alterations that are amenable to targeted therapy, offering the potential for individualized management strategies for the treatment of these aggressive tumors with different etiology. Discordant PD-L1 expression in the primary and metastatic vulvar SCCs highlights the importance of evaluation of PD-L1 expression in different locations to avoid false negative information provided for immunotherapy.

Purpose: Two viral oncoproteins, E6 and E7, are expressed in all human papillomavirus (HPV)–infected cells, from initial infection in the genital tract to metastatic cervical cancer. Intramuscular vaccination of women with high-grade cervical intraepithelial neoplasia (CIN2/3) twice with a naked DNA vaccine, pNGVL4a-sig/E7(detox)/HSP70, and a single boost with HPVE6/E7 recombinant vaccinia vaccine (TA-HPV) elicited systemic HPV-specific CD8 T-cell responses that could traffic to the lesion and was associated with regression in some patients (NCT00788164). Experimental Design: Here, we examine whether alteration of this vaccination regimen by administration of TA-HPV vaccination in the cervicovaginal tract, rather than intramuscular (IM) delivery, can more effectively recruit antigen-specific T cells in an orthotopic syngeneic mouse model of HPV16+ cervical cancer (TC-1 luc). Results: We found that pNGVL4a-sig/E7(detox)/HSP70 vaccination followed by cervicovaginal vaccination with TA-HPV increased accumulation of total and E7-specific CD8+ T cells in the cervicovaginal tract and better controlled E7-expressing cervicovaginal TC-1 luc tumor than IM administration of TA-HPV. Furthermore, the E7-specific CD8+ T cells in the cervicovaginal tract generated through the cervicovaginal route of vaccination expressed the α4β7 integrin and CCR9, which are necessary for the homing of the E7-specific CD8+ T cells to the cervicovaginal tract. Finally, we show that cervicovaginal vaccination with TA-HPV can induce potent local HPV-16 E7 antigen-specific CD8+ T-cell immune responses regardless of whether an HPV DNA vaccine priming vaccination was administered IM or within the cervicovaginal tract. Conclusions: Our results support future clinical translation using cervicovaginal TA-HPV vaccination. Clin Cancer Res; 22(3); 657–69. ©2015 AACR. See related commentary by Nizard et al., p. 530

BackgroundPeritoneal carcinomatosis is a hallmark of advanced peritoneal tumor progression, particularly for tubal/ovarian high-grade serous carcinomas (HGSCs). Patients with peritoneal carcinomatosis have poor survival rates and are difficult to treat clinically due to widespread tumor dissemination in the peritoneal cavity.MethodsWe developed a clinically relevant, genetically induced, peritoneal carcinomatosis model that recapitulates the histological morphology and immunosuppressive state of the tumor microenvironment of metastatic peritoneal HGSCs by intraperitoneally injecting shp53, AKT, c-Myc, luciferase and sleeping beauty transposase, followed by electroporation (EP) in the peritoneal cavity of immunocompetent mice (intraperitoneal (IP)/EP mice).ResultsSimilar to the spread of human ovarian cancers, IP/EP mice displayed multiple tumor nodules attached to the surface of the abdomen. Histopathological analysis indicated that these tumors were epithelial in origin. These IP/EP mice also displayed a loss of CD3+T cell infiltration in tumors, highly expressed inhibitory checkpoint molecules in tumor-infiltrating and global CD4+and CD8+T cells, and increased levels of transforming growth factor-β in the ascites, all of which contribute to the promotion of tumor growth.ConclusionsOverall, our tumor model recapitulates clinical peritoneal HGSC metastasis, which makes it ideal for preclinical drug screening, testing of immunotherapy-based therapeutics and studying of the tumor biology of peritoneal carcinomatosis.

A technology to prime desired populations of T cells in the body-particularly those that possess low avidity against target antigen-would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide-major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin V (ANXA5), which-in a concerted and synergistic manner-couples the early onset of TCR signaling by cognate pMHC with a surge in pMHC-TCR affinity, with repeated pMHC encounters, and with widespread TCR cross-linking. In our system, ANXA5 is linked to pMHC and firmly engages the plasma membrane of cognate T cells upon (and only upon) the early onset of TCR signaling. ANXA5, in turn, exerts a mechanical force that stabilizes interactions at the TCR-pMHC interface and facilitates repeated, serial pMHC encounters. Furthermore, ANXA5 quickly arranges into uniform 2D matrices, thereby prompting TCR cross-linking. Fusion of ANXA5 to pMHC augments lymphocyte activation by several orders of magnitude (>1,000-fold), bypasses the need for costimulation, and breaks tolerance against a model self-antigen in vivo. Our study opens the door to the application of synthetic, feedback-driven self-assembly platforms in immune modulation.

// Ruey-Shyang Soong 1, 5, 6 , Ravi K. Anchoori 4 , Benjamin Yang 1 , Andrew Yang 1 , Ssu-Hsueh Tseng 1 , Liangmei He 1 , Ya-Chea Tsai 1 , Richard B.S. Roden 1, 2, 4 , Chien-Fu Hung 1, 4 1 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 2 Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 3 Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 4 Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD, United States 5 Department of General Surgery, Chang Gung Memorial Hospital at Keelung, Keelung City, Taiwan 6 Department of Chang Gung University, College of Medicine, Taoyuan, Taiwan Correspondence to: Chien-Fu Hung, email: chung2@jhmi.edu Keywords: RPN13, proteasome, immunosuppression, MDSCs, Stat3 Received: January 14, 2016 Accepted: September 12, 2016 Published: September 17, 2016 ABSTRACT Myeloid-derived-suppressor cells (MDSCs) are key mediators of immune suppression in the ovarian tumor microenvironment. Modulation of MDSC function to relieve immunosuppression may enhance the immunologic clearance of tumors. The bis-benzylidine piperidone RA190 binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills ovarian cancer cells by triggering proteotoxic stress. Here we examine the effect of RA190 treatment on the immunosuppression induced by MDSCs in the tumor microenvironment, specifically on the immunosuppression induced by MDSCs. We show that RA190 reduces the expression of Stat3 and the levels of key immunosuppressive enzymes and cytokines arginase, iNOS, and IL-10 in MDSCs, while boosting expression of the immunostimulatory cytokine IL-12. Furthermore, we show that the RA190-treated MDSCs lost their capacity to suppress CD8+ T cell function. Finally, we show that RA190 treatment of mice bearing syngeneic ovarian tumor elicits potent CD8+ T cell antitumor immune responses and improves tumor control and survival. These data suggest the potential of RA190 for ovarian cancer treatment by both direct killing of tumor cells via proteasome inhibition and relief of MDSC-mediated suppression of CD8 T cell-dependent antitumor immunity elicited by the apoptotic tumor cells.

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8+T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)—together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen—elicited robust E7-specific CD8+T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.

Multimodality treatments that combine conventional cancer therapies with antigen-specific immunotherapy have emerged as promising approaches for the control of cancer. In the current study, we have explored the effect of doxorubicin on the antigen-specific immune responses generated in mice vaccinated with calreticulin (CRT)/E6 and/or Ii-PADRE DNA. We observed that pretreatment with doxorubicin suppressed the E6-specific CD8+ T-cell immune responses generated by CRT/E6 DNA vaccination in vaccinated mice. In contrast, pretreatment with doxorubicin enhanced the PADRE-specific CD4+ T-cell immune responses generated by Ii-PADRE DNA vaccination. Furthermore, coadministration of Ii-PADRE DNA could not only reverse the suppression, but also enhanced the E6-specific CD8+ T-cell responses in CRT/E6-vaccinated mice pretreated with doxorubicin. Finally, treatment with doxorubicin followed by CRT/E6 combined with Ii-PADRE DNA vaccination led to enhanced antitumor effects and prolonged survival in TC-1 tumor-bearing mice. The clinical implications of the current study are discussed.

DNA vaccination has emerged as a promising strategy for cancer immunotherapy. However, since DNA vaccines have low immunogenicity, various strategies have been developed to enhance the potency of DNA vaccines. In the current study, we aim to determine whether the potency of the DNA vaccine encoding human papillomavirus type 16 (HPV-16) E7 antigen can be enhanced by IL-2. We have generated a DNA vaccine encoding IL-2 linked to HPV-16 E7 antigen. Our results indicate that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the highest frequency of E7-specific CD8(+) T cells. We also found that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the strongest protective as well as therapeutic anti-tumor effect against E7-expressing tumors. In addition, it was observed that CD8(+) T cells were mainly responsible for the antitumor effect generated by the DNA vaccine encoding a fusion of IL-2 and E7 proteins. Thus, we conclude that the linkage of IL-2 to HPV-16 E7 antigen significantly enhances the DNA vaccine potency against E7-expressing tumors. Our strategy may potentially be used in other antigenic systems to control infectious diseases and/or cancer.

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8+ (CD8+) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8+ T cells through its interaction with the α4β1 integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with α4β1 integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC. [Cancer Res 2007;67(4):1832–41]

Mesothelin is highly expressed in a majority of ovarian cancer cells and is expressed at low levels in normal cells. Therefore, mesothelin represents a potential target antigen for ovarian cancer vaccine development. DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In the current study, we created a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. Our results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540-549 (pcDNA3-Hmeso540-beta2m-A2) generated strong human mesothelin peptide (aa540-549)-specific CD8+ T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3-Hmeso540-beta2m-A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540-549 represents a potential opportunity for the clinical translation of DNA vaccines against human mesothelin-expressing tumors, particularly ovarian cancer cells.

Background Human Papillomavirus is responsible for over 99 % of cervical cancers and is associated with cancers of the head and neck. The currently available prophylactic vaccines against HPV do not generate therapeutic effects against established HPV infections and associated lesions and disease. Thus, the need for a therapeutic vaccine capable of treating HPV-induced malignancies persists. Synthetic long peptides vaccination is a popular antigen delivery method because of its safety, stability, production feasibility, and its need to be processed by professional antigen presenting cells before it can be presented to cytotoxic CD8+ T lymphocytes. Cancers in the buccal mucosa have been shown to elicit cancer-related inflammations that are capable of recruiting inflammatory and immune cells to generate antitumor effects. In the current study, we evaluated the therapeutic potential of synthetic HPV long peptide vaccination in the absence of adjuvant in the TC-1 buccal tumor model. Result We show that intratumoral vaccination with E7 long peptide alone effectively controls buccal TC-1 tumors in mice. Furthermore, we observed an increase in systemic as well as local E7-specific CD8+ T cells in buccal tumor-bearing mice following the vaccination. Finally, we show that induction of immune responses against buccal tumors by intratumoral E7 long peptide vaccination is independent of CD4+ T cells, and that the phenomenon may be related to the unique environment associated with mucosal tissues. Conclusion Our results suggest the possibility for clinical translation of the administration of adjuvant free therapeutic long peptide vaccine as a potentially effective and safe strategy for mucosal HPV-associated tumor treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13578-016-0083-9) contains supplementary material, which is available to authorized users.

Vaccinia virus has been shown to efficiently infect tumor cells. Therefore, vaccinia virus represents a potentially safe and effective antitumor agent against ovarian cancer. Here, we assessed the ability of vaccinia virus to preferentially infect and control both human and murine ovarian tumors in vivo. We used the non-invasive luminescence imaging system to monitor the infection and suppression of ovarian tumors by vaccinia in live mice. Our data indicated that vaccinia was able to effectively infect and kill both human and murine ovarian tumors. Vaccinia virus administered to mice intraperitoneally was specifically targeted to the murine or human ovarian tumors and led to antitumor responses. These findings suggest that vaccinia virus is capable of selectively targeting and controlling ovarian tumors. Thus, intraperitoneal injection with vaccinia virus may provide a potentially effective strategy for treating advanced-stage ovarian cancers.

The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8+ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8+ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.

We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8+T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8+T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2Kb, is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8+T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.

Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract. Conclusions: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. Clin Cancer Res; 20(21); 5456–67. ©2014 AACR.