Therapeutic Antitumor Responses Research Articles

Vaccination Against Androgen Receptor Splice Variants to Immunologically Target Prostate Cancer.

Background/Objectives: Androgen receptor (AR) expression and signaling are critical for the progression of prostate cancer and have been the therapeutic focus of prostate cancer for over 50 years. While a variety of agents have been developed to target this axis, many of these fail due to the emergent expression of AR RNA splice variants, such as AR-V7, that can signal independently of ligand binding. Other therapies, such as vaccination against prostate-specific antigens, have achieved FDA approvals but have fallen short of being incorporated as standard-of-care therapies for advanced prostate cancer. This may be due to the elevated level of immunosuppression observed in prostate cancer, which remains largely refractory to immune checkpoint blockade. Methods: We developed a vaccine targeting AR-V7, a common isoform associated with treatment resistance, and demonstrated its ability to elicit AR-V7-specific immunity and enable anti-tumor responses against AR-V7+ cancers in subcutaneous tumor models. Results: Our studies also revealed that AR-V7 expression conferred an immune suppressive phenotype that was significant in a non-AR-dependent prostate cancer model. Notably, in this model, we found that vaccination in combination with enzalutamide, an AR antagonist, suppressed these aggressive immune suppressive cancers and resulted in enhanced survival in comparison to control vaccinated and enzalutamide-treated mice. While anti-PD-1 immune checkpoint inhibition (ICI) alone slowed tumor growth, the majority of vaccinated mice that received anti-PD-1 therapy showed complete tumor elimination. Conclusions: Collectively, these results validate the importance of AR signaling in prostate cancer immune suppression and suggest the potential of AR-V7-specific vaccines as therapeutic strategies against prostate cancer, offering significant protective and therapeutic anti-tumor responses, even in the presence of androgen signaling inhibitors.

Liposomal celecoxib combined with dendritic cell therapy enhances antitumor efficacy in melanoma.

Cancer vaccine efficacy is limited by the immunosuppressive nature of the tumor microenvironment created by inflammation, immune inhibitory factors, and regulatory T cells (Tregs). Inspired by the role of cyclooxygenase-2 (COX-2) in inflammation in the tumor site, we proposed that normalization of the tumor microenvironment by celecoxib as a COX-2 inhibitor might improve the efficacy of Dendritic Cell (DC) therapy in a melanoma model. In the present study, liposomal celecoxib (Lip-CLX) was combined with ex vivo generated DC vaccines pulsed with gp100 peptide (in liposomal and non-liposomal forms) for prophylactic and therapeutic evaluation in the B16F10 melanoma model. Tumor site analysis by flow cytometry demonstrated that intravenous administration of Lip-CLX at a dose of 1 mg/kg in four doses effectively normalized the tumor microenvironment by reducing Tregs and IL-10 production. Furthermore, in combination with DC vaccination (DC + Lip-peptide+Lip-CLX), it significantly increased tumor-infiltrating CD4+ and CD8+ T cells and secretion of IFN-γ. This combinatorial strategy produced an effective prophylactic and therapeutic antitumor response, which reduced tumor growth and prolonged the overall survival. In conclusion, our findings suggest that the liposomal celecoxib targets the inhibitory mechanisms of the tumor microenvironment and broadens the impact of DC therapy to improve the outcome of immunotherapy in solid tumors.

Protein corona-driven nanovaccines improve antigen intracellular release and immunotherapy efficacy.

During vaccine delivery in vivo, the vaccine carrier dynamically adsorbs the surrounding proteins or biomacromolecules to form a protein corona layer, which determines the physiological and therapeutic responses of the vaccine. Although the importance of the protein corona effect in drug delivery is widely accepted, understanding of the rational use of the protein corona to improve antigen controlled release is still sparse. Here, we constructed a protein corona-driven nanovaccine (PCNV), which has the dual effects of resisting the protein corona-induced antigen extracellular release and promoting protein corona-triggered antigen cytosolic release under reductive conditions. Specifically, the nanovaccine was formulated via the assembly of fluorinated dendrigraft-poly-lysine and cleavable antigen-CpG conjugate. Before entering antigen-presenting cells (APCs), the anchoring effect of CpG was used to avoid the dissociation of antigens from the carrier even under the protein corona effect. While nanovaccine enters the APCs, the intracellular reducing conditions can induce a break in the disulfide bond between CpG and antigen. Notably, at the same time, the intracellular protein corona effect triggers antigen release from the carrier and achieves efficient antigen presentation. In addition, the PCNV produced a significant prophylactic and therapeutic antitumor response in the mouse model. Therefore, the rational use of the protein corona effect provides an effective strategy for vaccine delivery.

Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Enhancing the anti-leukemia immunity of acute lymphocytic leukemia-derived exosome-based vaccine by downregulation of PD-L1 expression.

Cell-released nanovesicles can induce anti-leukemia immunity. Leukemia cell-derived exosomes (LEXs) are promising anti-tumor vaccine components for cancer immunotherapy. Nonetheless, LEX-based vaccines show modest potency in vivo, likely due to the presence of immunosuppressive PD-L1 proteins in the exosomes. We hypothesized that targeting exosomal PD-L1 could optimize LEX-based vaccines. To test this hypothesis, we compared the capacity of exosomes derived from PD-L1-silenced acute lymphocytic leukemia-derived leukemia cells (LEXPD-L1si) and non-modified exosomes to induce anti-leukemia immunity. Lentivirus-mediated PD-L1 shRNA was used to downregulate PD-L1 expression in parental leukemia cells and LEXs. LEXPD-L1si were characterized by electron microscopy, Western blotting, nanoparticle tracking analysis and flow cytometry, and their anti-leukemia immune effects were tested on immune cells and in animal models. In the present study, lentivirus-mediated PD-L1 shRNA successfully downregulated PD-L1 expression in parental leukemia cells and in LEXs. LEXPD-L1si induced better DC maturation and subsequently enhanced T cell activation, as compared with non-modified LEXs. Consistently, immunization with LEXPD-L1si induced greater T cell proliferation and Th1 cytokine release. LEXPD-L1si was a more potent inducer of antigen-specific cytotoxic lymphocyte (CTL) response. Finally, we vaccinated DBA/2 mice with exosome formulations to test their ability to induce both protective and therapeutic anti-tumor CTL responses in vivo. Vaccination with LEXPD-L1si strongly inhibited tumor growth and prolonged survival of immunized mice. Downregulation of exosomal PD-L1 expression in LEXs effectively induces more potent anti-leukemia immunity. Therefore, our strategy for optimizing LEX-based vaccine has a potential application in leukemia immunotherapy.

Alphavirus-Driven Interferon Gamma (IFNg) Expression Inhibits Tumor Growth in Orthotopic 4T1 Breast Cancer Model.

Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogram the tumor microenvironment; however, the antitumor immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumor potential in vitro in a model of 3D spheroids and in vivo in an immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived, IFN-g-stimulated bone marrow macrophages can be used to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoral administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumor growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intratumoral lymphoid cells isolated from tumors after SFV/IFNg treatment revealed increased CD4+ and CD8+ and decreased T-reg (CD4+/CD25+/FoxP3+) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38, and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumor T-cell response and inhibits myeloid cell infiltration in treated tumors.

Systemic effect of radiotherapy before or after nivolumab in lung cancer: an observational, retrospective, multicenter study.

The combination of radiotherapy (RT) and programmed death 1 inhibitors seems to increase antitumor immune responses. To assess the outcome and the role of the best combination sequence, i.e. immunotherapy given before, during, and/or after RT, in patients with non-small cell lung cancer (NSCLC). We conducted an observational, retrospective analysis of 95 consecutive patients with advanced NSCLC who received any radiotherapy treatment and nivolumab, as clinically indicated. Median overall survival (OS) and the 95% confidence interval (CI) were estimated with the Kaplan-Meier method. Cox model was used to obtain hazard ratio (HR) and associated 95% CI with statistical inference by log-rank statistic. Median OS was 11.9 months (95% CI, 6.6-17.2). Patients who received radiotherapy during an immune checkpoint inhibitor treatment started more than 60 days before showed a better outcome than patients who started immunotherapy over 60 days after RT ending (HR, 2.90 [1.37-6.12], p = 0.005; median OS, 22.4 months vs 8.6 months, p = 0.005). Median progression-free survival was 6.3 months (95% CI, 4.6-8.0). This study shows that combining irradiation with nivolumab for the treatment of advanced NSCLC leads to improved OS. The optimal time window for the combination of RT and immunotherapy seems to play a critical role for therapeutic antitumor response derived by abscopal effect.

Polymer-lipid hybrid nanovesicle-enabled combination of immunogenic chemotherapy and RNAi-mediated PD-L1 knockdown elicits antitumor immunity against melanoma

Immunotherapy has revolutionized cancer treatment; however, only a limited portion of patients show responses to currently available immunotherapy regimens. Here, we demonstrate that RNA interference (RNAi) combined with immunogenic chemotherapy can elicit potent antitumor immunity against melanoma. Specially, we developed cationic polymer-lipid hybrid nanovesicles (P/LNVs) as a new delivery system for doxorubicin and small interfering RNA (siRNA) with extensive cytotoxicity and gene silencing efficiency towards B16 cells. The deployment of doxorubicin-loaded P/LNVs augmented the expression and presentation of endogenous tumor antigens directly in situ by inducing the immunogenic cell death of B16 cells through poly(ADP-ribose) polymerase 1-dependent (PARP1) apoptosis pathway; thereby, eliciting remarkable antitumor immune responses in mice. Leveraging dying B16 cells as a vaccination strategy in combination with RNAi-based programmed cell death ligand 1 (PD-L1) knockdown showed efficacy in both prophylactic and metastasis melanoma settings. Strikingly, PD-L1 blockade synergized with a sub-therapeutic dose of doxorubicin triggered robust therapeutic antitumor T-cell responses and eradicated pre-established tumors in 30% of mice bearing B16 melanoma. Our findings indicated that this combination treatment provided a new powerful immunotherapy modality, characterized by markedly increased infiltration of effector CD8+ T cells and effective alleviation of the immunosuppressive microenvironment in tumors. P/LNVs is a versatile and highly scalable carrier that can enable a broad combination of nanomedicine and RNAi, providing new therapeutic strategies for advanced cancers.

NY-ESO-1 Protein Vaccine Combining Alum, CpG ODN, and HH2 Complex Adjuvant Induces Protective and Therapeutic Anti-Tumor Responses in Murine Multiple Myeloma.

BackgroundNY-ESO-1 is an ideal target for multiple myeloma immunotherapy. Alum, CpG ODN and HH2 complex is a safe and effective adjuvant for cancer vaccine.MethodsWe constructed NY-ESO-1 protein vaccine combined with alum, CpG ODN, and HH2 complex adjuvant to immunize the BALB/c mice inoculated with NS-1 murine multiple myeloma cells. Then, we determined the immunogenicity and anti-tumor effects in prophylactic and therapeutic models by analyzing the NY-ESO-1 antibody titer, evaluating IL4/INF-γ expression, and assessing cytotoxic T lymphocytes activities. The side-effects of vaccines were also evaluated.ResultsThe group of NY-ESO-1 protein vaccine combining alum, CpG ODN, and HH2 complex adjuvant is more capable of stimulating both humoral and cellular tumor-specific immune responses to prolong the survival of the mice and inhibit tumor growth in prophylactic and therapeutic immunotherapy. The marked side-effects were not detected in immunized mice.DiscussionThe results suggest that alum, CpG ODN, and HH2 complex as a novel immune adjuvant combined cancer vaccine could improve the immunity efficiency in a murine multiple myeloma model.

Microbial regulation of tumor development and responses to tumor therapy

Viruses and bacteria play central roles in gastrointestinal tumor development. This includes well-characterized contributions of Helicobacter pylori to gastric carcinoma and MALT lymphoma and of Hepatitis B and C virus infections to hepatocellular cancer. However, recent studies have demonstrated a much broader role of the microbiota in the regulation of cancer development. As such, it was shown that barrier defects and alterations in microbial community structure contribute to colorectal and hepatocellular cancer. Moreover, intriguing studies have highlighted the microbiota as a central regulator of the efficacy of systemic anti-tumor therapies. Here, we provide an overview of recent observations on the role of the microbiota in tumor development and the regulation of therapeutic anti-tumor responses and discuss the implications of these findings for clinical diagnostics and treatment.

Abstract A034: High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens

Abstract The growing use of immunotherapy to treat advanced cancers has brought about a revolution in techniques to mobilize the immune system to antitumor effect. Chimeric antigen receptor (CAR) T-cells targeting CD19 constitute the first modified T-cell products to garner FDA approval for clinical use. However, CAR technologies can only targeT-cell surface antigens, representing approximately one quarter of potential targets. In contrast, T-cell receptor (TCR) therapies can target peptides presented by the major histocompatibility complex (MHC), including those derived from intracellular antigens. Hitherto, such receptors have generally been identified through low-throughput techniques using cancer patients’ blood, followed by affinity-maturation of TCRs, a step that can decrease safety. Here, we demonstrate a novel pipeline for the identification of potential therapeutic TCRs from the naïve repertoire of healthy individuals. Using a technique called Multiplexed Identification of T-cell Receptor Antigen specificity (MIRA), we input hundreds of antigens of interest, including tumor-associated antigens, viral antigens and neoantigens, and identify thousands of TCRs to these antigens. These TCRs then undergo evaluation for affinity, avidity, cytokine release, cytotoxicity and safety. Cytotoxicity is demonstrated using both peptide-loaded and endogenously presented peptides. Safety is evaluated using alanine-glycine scans; evaluation of reactivity of TCRs againsT-cell lines and primary cells is planned. We have fully characterized several TCRs targeting clinically relevant targets, which demonstrated improved avidity and cytolysis relative to a benchmark TCR, and a promising preliminary safety profile. In the recent year, cancer neoantigens as targets for natural and therapeutic antitumor responses have also gained momentum given their attractive product profile. Hereto, we also show data for TCRs against shared neoantigens. Citation Format: Mark Klinger, Peter Ebert, Edward Osborne, Ruth Taniguchi, Joyce Hu, Tim Hayes, Sharon Benzeno, Adria Carbo, Melanie Laur, Erica Eggers, Harlan Robins. High-throughput identification of naturally occurring T-cell receptors with therapeutic potential against tumor-associated, viral and neoantigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A034.

A Synthetic DNA, Multi-Neoantigen Vaccine Drives Predominately MHC Class I CD8+ T-cell Responses, Impacting Tumor Challenge.

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

The novel agonistic iNKT-cell antibody NKT14m induces a therapeutic antitumor response against B-cell lymphoma

ABSTRACTInvariant natural killer T (iNKT) cells are a small population of T lymphocytes that expresses an invariant T cell receptor with a unique specificity for glycolipid antigens. Their activation using the glycolipid α-galactosylceramide (α-GalCer) triggers innate and adaptive immune responses. The use of α-GalCer in preclinical models as a single antitumor treatment showed moderate effect, but its efficacy in cancer patients was less effective. In addition, this glycolipid induces long-term iNKT-cell anergy precluding the possibility of retreatment. Recently, the first murine iNKT-cell agonistic antibody, NKT14m, has been developed. Here, we analyzed, for the first time, the antitumor efficacy of NKT14m in a B-cell lymphoma model. In a therapeutic setting, a single dose of NKT14m had a moderate antitumor efficacy that was associated with an increase of IFN-γ producing iNKT cells even after a second dose of the NKT14m antibody. Importantly, the combination of a single dose of NKT14m with cyclophosphamide had a potent antitumor efficacy and long-lasting immunity in vivo. Our findings provide the first evidence of the in vivo antitumor efficacy of NKT14m antibody, showing that, either alone or in combination with chemotherapy, induces an effective antitumor response. These results open new opportunities for iNKT-cell mediated immunotherapy to treat B-cell lymphoma.

Abstract 1733: Wnt antagonists synergize with immune checkpoint inhibitors to enhance anti-tumor responses

Abstract Aberrant Wnt signaling through overexpression or activating mutations in Wnt pathway proteins is responsible for the initiation and progression of numerous cancers. While enhanced Wnt signaling has been shown to play a major role in cancer stem cell biology, more recent studies have implicated Wnt in the development of resistance to anti-tumor immune responses. Here, we show that in various syngeneic murine tumor models, targeting Wnt signaling using the Fzd receptor monoclonal antagonist antibody, OMP-18R5 (vantictumab) or the pan-Wnt decoy receptor Fc fusion protein OMP-Fzd8-Fc (ipafricept) in combination with immune checkpoint inhibitors anti-CTLA-4 or anti-PD1 induce synergistic anti-tumor responses leading to decreased tumor volume and increased infiltration of activated CD8+ T cells into the tumor microenvironment. In addition, we show that combined Wnt and immune checkpoint inhibition decrease regulatory T cells (Tregs), enhance cytotoxic T cell activity and increase antigen presentation by APCs. More importantly, we demonstrate that anti-PD1 and the Wnt antagonists decrease immune suppressive myeloid cell populations, which may enhance therapeutic efficacy and anti-tumor responses. Therefore, these results suggest that co-targeting Wnt and immune checkpoint proteins may provide valuable opportunities for novel combination strategies for immunotherapeutic clinical development. Citation Format: Jenny Ross, Timothy Hoey, Ann Kapoun, John Lewicki, Austin Gurney, Christopher L. Murriel. Wnt antagonists synergize with immune checkpoint inhibitors to enhance anti-tumor responses [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 1733.

Irradiation enhances dendritic cell potential antitumor activity by inducing tumor cell expressing TNF-α.

Dendritic cells (DCs)-based tumor vaccines have shown to be the promising methods for inducing therapeutic antitumor response. However, DCs alone rarely carry curative antitumor activity, and the immunosuppressive microenvironment may contribute to this defect of DC vaccinal function. Irradiation in combination with DCs has been shown to promote immune-mediated tumor destruction in preclinical studies. However, little is known about how irradiation alters the tumor microenvironment, and what host pathways modulate the activity of administrated DCs. In this study, BALB/c mice and the 4T1 breast cancer cell line were used in a tumor-bearing model. The tumor-bearing mice were irradiated locally up to 10Gy for 3 consecutive days or a single dose of 30Gy using a cesium source. Studies of dynamic change of the tumor microenvironment in irradiated versus untreated tumors revealed that there was no obvious change on IL-10, IL-6 and TGF-β expression or production, whereas increased TNF-α level within the first 2weeks of irradiation. The increased TNF-α level is exactly right timing window for DCs injection, corresponding to the significant elevation of intratumoral CD8+ T infiltration and the regression of tumor size. With attention to scheduling, combination X-ray with DCs i.t. injection may offer a practical strategy to improve treatment outcomes.

GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8+ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8+ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.

DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect.

Cellular immune responses play a critical role in the eradication of intracellular infections and malignant cells through the recognition and subsequent removal of the infection or malignant cells. Effective antigen presentation is crucial for stimulating the immune system against malignant cells. Calreticulin (CRT) has been used to improve antigen presentation. However, CRT overexpression has been previously associated with the development of pancreatic and breast cancer. The import and retention signals of CRT in the endoplasmic reticulum (ER) can be used to overcome CRT overexpression. The present study describes the potent antitumor effect of a DNA vaccine encoding human papillomavirus type 16 E6 and E7 antigens flanked by ER import and retention signals (SP-E6E7m-KDEL). The effect of this vaccine was compared with that of E6 and E7 antigens fused to human full-length CRT (hCRT-E6E7m). In the present study, the effectiveness of SP-E6E7m-KDEL for inducing an interferon-γ antigen-specific, response and its therapeutic effect against tumors was demonstrated, which was as effective as immunization against those antigens fused to CRT. This simplified strategy, using ER import and retention signal peptides to direct antigens to this organelle, provides an efficient alternative to traditional vaccines and, more importantly, a safe and potent system to induce a therapeutic antitumor response.

Old-School Chemotherapy in Immunotherapeutic Combination in Cancer, A Low-cost Drug Repurposed.

Cancer immunotherapy has proven to be a potent treatment modality. Although often successful in generating antitumor immune responses, cancer immunotherapy is frequently hindered by tumor immune-escape mechanisms. Among immunosuppressive strategies within the tumor microenvironment, suppressive immune regulatory cells play a key role in promoting tumor progression through inhibiting the effector arm of the immune response. Targeting these suppressive cells can greatly enhance antitumor immune therapies, hence augmenting a highly effective therapeutic antitumor response. Several approaches are being tested to enhance the effector arm of the immune system while simultaneously inhibiting the suppressor arm. Some of these approaches are none other than traditional drugs repurposed as immune modulators. Cyclophosphamide, an old-school chemotherapeutic agent used across a wide range of malignancies, was found to be a potent immune modulator that targets suppressive regulatory immune cells within the tumor microenvironment while enhancing effector cells. Preclinical and clinical findings have confirmed the ability of low doses of cyclophosphamide to selectively deplete regulatory T cells while enhancing effector and memory cytotoxic T cells within the tumor microenvironment. These immune effects translate to suppressed tumor growth and enhanced survival, evidence of antitumor therapeutic efficacy. This article discusses the reincarnation of cyclophosphamide as an immune modulator that augments novel immunotherapeutic approaches. Cancer Immunol Res; 4(5); 377-82. ©2016 AACR.

A novel dendritic cell targeting HPV16 E7 synthetic vaccine in combination with PD-L1 blockade elicits therapeutic antitumor immunity in mice

ABSTRACTHuman papilliomavirus (HPV) oncogene E7, essential for the transformation and maintenance of the malignancy of cervical cancer cells, represents an ideal tumor-specific antigen for vaccine development. However, due to the poor immunogenicity of E7 protein, an effective therapeutic E7 vaccine is still lacking. Dendritic cells (DCs) are probably the most potent antigen presenting cells for the induction of cytotoxic T lymphocyte (CTL) response, which is crucial for tumor control. In this study, we tested whether targeting the E7 antigen to DCs in vivo would elicit therapeutic antitumor CTL response. We generated the DEC205-specific single-chain variable fragment (scFv) and E7 long peptide fusion protein [scFv(DEC205)-E7] based on the novel method of protein assembly we recently developed. This fusion protein vaccine demonstrated highly efficient DC-targeting in vivo and elicited much stronger protective CTL response than non-DC-targeting control vaccine in naive mice. Furthermore, the scFv(DEC205)-E7 vaccine showed significant therapeutic antitumor response in TC-1 tumor bearing mice. Importantly, PD-L1 blockade further improved the therapeutic effect of the scFv(DEC205)-E7 vaccine. Thus, the current study suggests an efficient strategy for cervical cancer immunotherapy by combining the DC(DEC205)-targeting E7 vaccine and PD-L1 blockade.

Abstract B183: Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment

Abstract The recent clinical successes of CTLA4 and PD-1 blockade have clearly demonstrated the therapeutic efficacy to enhance anticancer immune responses. Novel targets of immunomodulation are now under investigation to increase the number of immunotherapeutic options available for relapsing/refractory patients. Activation of co-stimulatory molecules with agonist monoclonal antibody (mAbs) represents the most rationale alternative option. Based on the impressive preclinical activity of stimulating the glucocorticoid-induced TNF receptor (GITR) with the mAb DTA-1, an agonist anti-human GITR mAb has very recently entered the clinical evaluation. Although DTA-1, as a monotherapy, proved effective in curing established immunogenic tumors, it failed against advanced poorly immunogenic models, such as B16 melanoma. As anti-GITR therapy is translated into clinic, defining the basis of its activity and failure is key to identify prognostic markers and more effective combination therapies. We thus studied how GITR stimulation with the agonist antibody DTA-1 affects T cells in B16 melanoma-bearing mice treated in curative (early) or refractory (advanced) condition, and verified their functional changes in vitro. In vivo, the comparison of advanced versus early B16 microenvironment before treatment revealed a significant increase in Treg frequency, which caused the Teff/Treg ratio to decrease, and a dysfunctional Teff phenotype. Moreover, B16 cells, in the setting of established tumors, expressed lower levels of MHC-I and MHC-II molecules. DTA-1 consistently reduced intratumor Treg frequency and expression of suppression markers in both curative and refractory conditions. However, Teff activation and tumor-associated MHCs were induced only when DTA-1 was administered in curative conditions. In vitro, pre-incubation of Tregs with DTA-1 completely abolished their ability to inhibit proliferation of target Teff, without affecting Treg expression of the lineage markers Foxp3 and Helios. In addition, treatment with DTA-1, in comparison with the matched IgG isotype control, boosted the release of IFN-gamma, TNF-alpha, IL-17 and IL-6 cytokines in cultures of Tregs, pointing to the promotion of a Th1/Th17 differentiation profile following GITR engagement. Interestingly, when B16 cells were added in culture with Tregs and incubated with DTA-1, they up-regulated MHC-I and MHC-II molecules in a Treg and/or DTA-1 dependent manner. The presence of tumor cells during exposure of Tregs to anti-GITR stimulation did not further alter their function and phenotype, indicating a major direct effect of DTA-1 on Tregs. To confirm the functional modulation of GITR stimulation in human Tregs, we tested their activity in vitro upon pre-incubation with a recombinant human GITR ligand (GITRL). We consistently found that proliferation of activated Teff was no longer suppressed if Tregs were previously treated with GITRL. These results validate GITR as a target to revert immune tolerance in the human setting. Our data indicate that anti-GITR therapy counteracts Treg-mediated immunosuppression independently from the stage of the disease, whereas T-cell activation and tumor immune recognition are favored selectively in the presence of low tumor burden. These findings suggest that, in the setting of advanced and poorly immunogenic tumors, anti-GITR therapy should be exploited in combination with treatments promoting Teff functions and/or tumor killing. Citation Format: Roberta Zappasodi, Sadna Budhu, Cailian Liu, Kyle Draleau, David Schaer, Hong Zhong, Xia Yang, Taha Merghoub, Jedd D. Wolchok. Treg inhibition by anti-GITR therapy evokes therapeutic antitumor responses selectively in the context of a pro-inflammatory tumor microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B183.