Local Antitumor Immunity Research Articles

Inhibition of melanoma by survivin-specific lymphocytes combined with CCL17 and granulocyte-macrophage colony-stimulating factor in a mouse syngeneic model.

As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better.

Radiation induces dynamic changes to the T cell repertoire in renal cell carcinoma patients

Clinical studies combining radiation and immunotherapy have shown promising response rates, strengthening efforts to sensitize tumors to immune-mediated attack. Thus, there is an ongoing surge in trials using preconditioning regimens with immunotherapy. Yet, due to the scarcity of resected tumors treated in situ with radiotherapy, there has been little investigation of radiation's sole contributions to local and systemic antitumor immunity in patients. Without this access, translational studies have been limited to evaluating circulating immune subsets and systemic remodeling of peripheral T cell receptor repertoires. This constraint has left gaps in how radiation impacts intratumoral responses and whether tumor-resident T cell clones are amplified following treatment. Therefore, to interrogate the immune impact of radiation on the tumor microenvironment and test the hypothesis that radiation initiates local and systemic expansion of tumor-resident clones, we analyzed renal cell carcinomas from patients treated with stereotactic body radiation therapy. Transcriptomic comparisons were evaluated by bulk RNA sequencing. T cell receptor sequencing monitored repertoires during treatment. Pathway analysis showed radiation-specific enrichment of immune-related processes, and T cell receptor sequencing revealed increased clonality in radiation-treated tumors. The frequency of identified, tumor-enriched clonotypes was tracked across serial blood samples. We observed increased abundance of tumor-enriched clonotypes at 2 wk postradiation compared with pretreatment levels; however, this expansion was not sustained, and levels contracted toward baseline by 4 wk posttreatment. Taken together, these results indicate robust intratumoral immune remodeling and a window of tumor-resident T cell expansion following radiation that may be leveraged for the rational design of combinatorial strategies.

Targeted Delivery of CXCL9 and OX40L by Mesenchymal Stem Cells Elicits Potent Antitumor Immunity

Major obstacles in immunotherapies include toxicities associated with systemic administration of therapeutic agents, as well as low tumor lymphocyte infiltration that hampers the efficacies. In this study, we report a mesenchymal stem cell (MSC)-based immunotherapeutic strategy in which MSCs specifically deliver T/natural killer (NK) cell-targeting chemokine CXCL9 and immunostimulatory factor OX40 ligand (OX40L)/tumor necrosis factor superfamily member 4 (TNFSF4) to tumor sites in syngeneic subcutaneous and azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced spontaneous colon cancer mouse models. This approach generated potent local antitumor immunity by increasing the ratios of tumor-infiltrating CD8+ T and NK cells and production of antitumor cytokines and cytolytic proteins in the tumor microenvironment. Moreover, it improved the efficacy of programmed death-1 (PD-1) blockade in a syngeneic mouse model and significantly suppressed the growth of major histocompatibility complex class I (MHC class I)-deficient tumors. Our MSC-based immunotherapeutic strategy simultaneously recruits and activates immune effector cells at the tumor site, thus overcoming the problems with toxicities of systemic therapeutic agents and low lymphocyte infiltration of solid tumors.

Active Microneedle Administration of Plant Virus Nanoparticles for Cancer in situ Vaccination Improves Immunotherapeutic Efficacy.

The solid tumor microenvironment (TME) poses a significant structural and biochemical barrier to immunotherapeutic agents. To address the limitations of tumor penetration and distribution, and to enhance antitumor efficacy of immunotherapeutics, we present here an autonomous active microneedle (MN) system for the direct intratumoral (IT) delivery of a potent immunoadjuvant, cowpea mosaic virus nanoparticles (CPMV) in vivo. In this active delivery system, magnesium (Mg) microparticles embedded into active MNs react with the interstitial fluid in the TME, generating a propulsive force to drive the nanoparticle payload into the tumor. Active delivery of CPMV payload into B16F10 melanomas in vivo demonstrated substantially more pronounced tumor regression and prolonged survival of tumor-bearing mice compared to that of passive MNs and conventional needle injection. Active MN administration of CPMV also enhanced local innate and systemic adaptive antitumor immunity. Our approach represents an elaboration of conventional CPMV in situ vaccination, highlighting substantial immune-mediated antitumor effects and improved therapeutic efficacy that can be achieved through an active and autonomous delivery system-mediated CPMV in situ vaccination.

Abstract IA17: PARP inhibition modulates the tumor immune microenvironment in Brca1-deficient ovarian tumor

Abstract Poly ADP ribose polymerase (PARP) inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We recently reported that inhibition with a PARP inhibitor (olaparib) triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. However, all PBM-bearing mice treated with olaparib alone or in combination with PD-1 blockade eventually succumbed to the disease. These data indicate that although multiple PARP inhibitors have been approved for the treatment of BRCA-deficient ovarian cancers, eradication of this type of cancer remains a challenge. I will discuss our recent efforts on identifying resistance mechanisms to PARP inhibition and developing potential therapeutic strategy that overcomes the resistance to improve the treatment outcome. Citation Format: Liya Ding, Qiwei Wang, Michael Kearns, Tao Jiang, Panagiotis Konstantinopoulos, Ursula Matulonis, Jean Zhao. PARP inhibition modulates the tumor immune microenvironment in Brca1-deficient ovarian tumor [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA17.

Radiation-Induced Immunity in Kidney Cancer Patients: Seq-ing Commonality Between Peripheral and Intratumoral T cell Repertoires

Abstract Radiation has long been part of the standard of care for cancer treatment. Clinical studies combining immunotherapy with radiation have shown promising response rates implicating radiotherapy as an inducer of tumor immunity. Due to the scarcity of human samples treated in situ with radiation, exploration of this phenomenon has been largely limited to murine models. While these data draw connections between intratumoral immune responses and wider effects in peripheral blood, advances in patient care are restricted by inherent differences between murine models and human disease. Therefore, to identify commonality between local intratumoral and systemic responses to radiation, we analyzed clinical trial samples from renal cell carcinoma (RCC) patients treated with stereotactic body radiation therapy (SBRT) at the primary tumor. With access to patient tumors and peripheral blood, we hypothesized that SBRT initiates local and systemic anti-tumor immunity. Pathway analysis from tumor RNAseq data showed SBRT-specific immune processes: TCR activation, increased costimulation, and chemokine signaling. Deeper examination through single cell RNA sequencing identified the sources of these signals. In parallel, analysis by T cell receptor sequencing (TCRseq) showed increased tumor T cell clonality with SBRT treatment. TCRseq of serial blood samples revealed tumor-enriched T cell clonotypes reaching peak abundance 2 weeks post-SBRT. This expansion was not sustained and contracted towards baseline levels 4 weeks after SBRT. Collectively, these results indicate a critical interval following radiation, wherein tumor-enriched T cell clones are expanded in circulation, that could be utilized to bolster responses to immunotherapy.

Combined CD44- and CD25-Targeted Near-Infrared Photoimmunotherapy Selectively Kills Cancer and Regulatory T Cells in Syngeneic Mouse Cancer Models.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a mAb conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is a surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, CD25-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Treg), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44- and CD25-targeted NIR-PIT also resulted in some complete remissions. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

Abstract P5-04-09: Deep phenotyping using CyTOF identifies peripheral blood immune signatures associated with clinical outcomes and molecular subtypes in patients with early-stage triple negative breast cancer (TNBC)

Abstract Background: Local antitumor immunity—as assessed by quantifying tumor-infiltrating immune cells—is increasingly recognized as a critical factor influencing prognosis and therapy response in TNBC. However, an understanding of systemic antitumor immune responses occurring in peripheral blood, and their influence on prognosis and chemotherapy response has not been rigorously studied. Methods: Cytometry by time-of-flight (CyTOFTM, Fluidigm) was used to examine viably cryopreserved peripheral blood mononuclear cell (PBMC) suspensions prospectively collected from patients with early-stage TNBC prior to initiation of standard neoadjuvant paclitaxel followed by doxorubicin and cyclophosphamide (NACT) as part of the BEAUTY study [1]. Samples were stained using a panel of metal-tagged antibodies, recognizing 30 surface proteins optimized for immune monitoring of human peripheral blood. Differential abundance analysis of immune cell subsets was carried out to evaluate differences between patients who achieved pCR versus those with residual disease after NACT, and between patients with known luminal androgen receptor (LAR) versus basal TNBC subtypes defined by bulk tumor RNA sequencing. Results: Viably cryopreserved PBMC samples from 40 treatment-naive TNBC patients were available for analysis. The median age was 52 years (range 32 - 73), with 6 (15%) patients having tumors classified as LAR TNBC, and the remaining 34 (85%) as basal TNBC. Overall, 21 (53%) patients achieved pCR after NACT. After acquisition on the mass cytometer, the median yield per sample was 626,815 single-cell events (range 42,786 - 1,035,575), with a median percent debris of 13.7% (range 14 - 58). Across the 40 PBMC samples, the total yield was 23,507,094 single-cell events. The median frequencies of major circulating immune cell subsets across the 40 TNBC patients were: T cells 53.9% (range 25.4 - 71.3), with 33.4% CD4+ T cells (range 11.4 - 46.7) and 10.3% CD8+ T cells (range 5.8 - 19.9); B cells 10.8% (3.3 - 32.6), NK cells 8.6% (1.7 - 17.0) and monocytes 10.6% (2.7 - 29.8). Examining pre-treatment blood samples, patients with residual disease after NACT exhibited a higher median frequency of baseline CD14+CD16- classical monocytes (7.5% vs. 4.1%, p=0.025) and a lower frequency of terminally-differentiated effector memory cytotoxic (CD8+) T cells (0.6% vs. 1.7%, p=0.038) compared to patients who achieved pCR. Patients with LAR TNBC also exhibited a higher frequency of CD14+CD16- classical monocytes (11.5% vs 4.3%, p=0.058), and in addition exhibited a lower frequency of central memory CD4+ T cells (10.4% vs 15.2%, p=0.048). No difference in CD8+ T cells was seen by LAR status. Additional associations of peripheral blood immune cell subsets and classic tumor pathological features will be presented at the meeting. Conclusion: To our knowledge, this is the first study focused on TNBC to demonstrate variation in peripheral blood immune cell populations by molecular TNBC subtype (LAR vs. basal), and by chemotherapy response. A higher frequency of circulating classical monocytes—which can infiltrate into tissues and give rise to macrophages—appears to be detrimental; whereas a higher frequency of circulating antigen-experienced memory CD8+ T cells seems to be protective, suggesting a putative role of this cell subset in TNBC anti-tumoral immunity. Reference: [1] Goetz MP et al. JNCI 2017, PMID:28376176 Citation Format: Roberto A Leon-Ferre, Kaitlyn McGrath, Jodi M Carter, Krishna R Kalari, Vera J Suman, Richard Weinshilboum, Liewei Wang, Keith L Knutson, Stephen M Ansell, Judy C Boughey, J C Villasboas, Matthew P Goetz. Deep phenotyping using CyTOF identifies peripheral blood immune signatures associated with clinical outcomes and molecular subtypes in patients with early-stage triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-09.

Repurposing the yellow fever vaccine for intratumoral immunotherapy

Live 17D is widely used as a prophylactic vaccine strain for yellow fever virus that induces potent neutralizing humoral and cellular immunity against the wild‐type pathogen. 17D replicates and kills mouse and human tumor cell lines but not non‐transformed human cells. Intratumoral injections with viable 17D markedly delay transplanted tumor progression in a CD8 T‐cell‐dependent manner. In mice bearing bilateral tumors in which only one is intratumorally injected, contralateral therapeutic effects are observed consistent with more prominent CD8 T‐cell infiltrates and a treatment‐related reduction of Tregs. Additive efficacy effects were observed upon co‐treatment with intratumoral 17D and systemic anti‐CD137 and anti‐PD‐1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized with 17D, intratumoral 17D treatment achieved better local and distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8 T‐cell infiltration in the treated tumor. The repurposed use of a GMP‐grade vaccine to be given via the intratumoral route in prevaccinated patients constitutes a clinically feasible and safe immunotherapy approach.

Abstract 1474: Repurposing the yellow fever vaccine for intratumoral immunotherapy

Abstract Introduction: Yellow Fever attenuated virus vaccine (17D) induces very potent cellular and humoral immunity. Its delivery in situ in the tumor bed may induce an immune response leading to the killing of infected cells. Experimental procedures: 17D virus was propagated in Vero cells and infectious supernatants were harvested. To evaluate in vitro sensitivity of tumors to 17D infection, a panel of human and mouse tumor cell lines were infected with increasing MOIs and viability was quantified. To evaluate the antitumor efficacy of intratumor (i.t.) injection of 17D, 4x106pfu/50μl were injected (4 doses) in MC38- and in B16-OVA-tumor bearing C57BL/6 mice. Systemic antitumor effects of i.t 17D were examined in bilateral MC38 models in which one of the tumors was left uninjected. Depletion experiments were performed to assess the contribution of CD4, CD8, Gr1 and NK cells. To determine the role of conventional type-I dendritic cells (cDC1), identical experiments were performed in BATF3-/- mice. Tumor and lymph node immune infiltrates were examined by multiparametric flow cytometry. Combination with intraperitoneal anti-PD1 or anti-CD137 was performed to improve antitumor efficacy. To evaluate the effects of 17D-preexisting immunity, mice were preimmunized with 17D prior to MC38 bilateral inoculation and were subsequently treated. To assess the individual cell type contribution to the antitumor effects induced upon 17D pre-immunization, CD8, CD4, CD8+CD4 or sera from preimmunized mice were passively transferred 24h prior to first 17D i.t injection to MC38-tumor bearing mice. Results: 17D live attenuated strain is able to induce cell death of mouse and human tumor cell lines. In MC38 or B16-OVA mice models, repeated i.t. injections of 17D clearly delayed tumor progression. Regarding the systemic effect of i.t. 17D, certain contralateral therapeutic efficacy was induced upon i.t injection in bilateral MC38 models. These local and distant effects were dependent on CD8+ and cDC1 cells. 17D i.t injection increased in CD8+ T cell infiltrates and decreased in Tregs in injected tumors, while in contralateral tumors induced an increase of CD8+ and NK cells expressing key immunomodulatory receptors. I.t. 17D with anti-CD137 monoclonal antibody (mAb) induced a strong synergistic effect, while combination with anti-PD-1 mAb showed mild local additive efficacy compared to 17D monotherapy. Both combinatorial treatments delayed contralateral tumor growth. Notably, 17D preimmunization induced an important improvement of local and distant antitumor immunity. Such beneficial effects of preimmunization were transferred along with CD8+ T cells. Conclusions: 17D can be safely injected i.t. in preclinical models, giving rise to immune-mediate antitumor effects that can be combined with other immunotherapy agents. The repurposed use of this GMP-grade vaccine in preimmunized patients represents a promising and feasible immunotherapy approach. Citation Format: M. Angela Aznar, Carmen Molina, Alvaro Teijeira, Arantza Azpilikueta, Saray Garasa, Alfonso R. Sanchez-Paulete, Iñaki Etxeberria, Maite Alvarez, Pedro Berraondo, Ignacio Melero. Repurposing the yellow fever vaccine for intratumoral immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1474.

Abstract 3179: Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC which is among the 10 most common cancers in both men and women. Chimeric Antigen Receptor (CAR) T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR T cells, the suppressive tumor microenvironment, and on-target off-tumor toxicities resulted from the sharing of CAR T targeting epitopes on healthy tissues. To combat the suppressive microenvironment, immune checkpoint blockade has shown promising effect on antitumor response by restoring the local antitumor immunity. CAR T cell factories were designed to empower CAR T cells through the secretion of human anti-immune checkpoint inhibitor monoclonal antibodies (mAbs) locally at the tumor site. Our results show a dramatic improvement in CAR T killing of ccRCC in vitro and in vivoby reversing CAR T cell and tumor infiltrating lymphocyte (TIL) exhaustion. CAIX is an ideal target for ccRCC therapy and used as a CAR target for the first clinical trial. However, it led to adverse side effects (ADEs) due to CAIX expression on the bile duct. Therefore, it is crucial to develop a CAR with elevated efficacy and safety (limited on-target off-tumor effect). To achieve that, the 2nd targeting scFv was introduced in the CAR T cell factory together with anti-CAIX scFv to enable the CAR to target two unique antigens simultaneously. By IHC staining of ccRCC patient samples, we found that target B is an ideal target to be utilized as the 2nd target since it is highly expressed on ccRCC and co-expressed with CAIX. Our 27 billion-member human scFv-phage display library was panned against the antigen expressing skrc-59 ccRCC cells to identify novel scFvs. Their binding kinetics (Kon/Koff) were then measured and scFvs with desirable kinetics were evaluated for their ability to bind with antigen expressing cells. Ideal candidates were cloned into vectors where anti-target B and anti-CAIX scFvs were combined in different permutations by changing the order of the two targeting scFvs with various linkers connected to a costimulatory domain (CD28, 41BB) and an activating domain (CD3). Primary T cells isolated from PBMCs were transduced to express dual CAR and were tested against different cell lines in vitro. For further evaluation in vivo, a humanized orthotopic ccRCC mouse model was established by injecting luciferized ccRCC cells under the kidney capsule of NSG-SGM3 mice with a reconstituted human immune system. In summary, utilizing a dual CAR T discovery platform, we generated a series of CARs with different scFvs, linkers, and hinges. By combining the best dual CAR with immune checkpoint blockade as payload, we propose that lead 2nd generation CAR T cell factory candidates will be discovered that should mitigate against ADEs on normal tissues and hold great promise for the treatment of ccRCC. Citation Format: Yufei Wang, Eloah Rabello Suarez, Matthew Chang, Rebecca Jennings, Sabina Signoretti, Quan Zhu, Wayne Marasco. Design and activity of 2nd generation, dual-targeted CAR T cell factories against ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3179.

Immune landscape of neoadjuvant/preoperative chemoradiotherapy in pancreatic cancer.

e15709 Background: Neoadjuvant/preoperative chemoradiotherapy in pancreatic cancer has been implicated to induce a local anti-tumor immunity, however the molecular mechanism remains a conundrum. Methods: Two patients’ cohort samples with/without preoperative chemoradiotherapy were used (n = 26 and n = 20, respectively). The treatment-driven immune microenvironment changes were analyzed by stratified bioinformatic approaches i.e. heat map analysis of immune-related genes selected by Gene Ontology, GSEA with Immunologic Signature data base, and Ingenuity Pathway Analysis (IPA). The results were further validated by immunohistochemical approaches. Results: Preoperative treatment induced 212 genes including DC-SIGN (CD209) and 13 immune-associated pathways such as TCR signaling, with an increasing shift of CD8 T-cell population by upregulation of MAL (T-cell differentiation protein). Consistently, immunohistochemical findings showed that the accumulation of DC-SIGN (+) dendritic cells and MAL (+) lymphocytes were significantly increased in patients with preoperative therapy. Conclusions: Preoperative chemoradiotherapy induces a shift toward CD8 T-cell generation mediated by DC-SIGN (+) dendritic cells and MAL (+) lymphocytes, leading to enhance anti-tumor immunity.

Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called “hot” tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

Illuminating the Numbers: Integrating Mathematical Models to Optimize Photomedicine Dosimetry and Combination Therapies.

Cancer photomedicine offers unique mechanisms for inducing local tumor damage with the potential to stimulate local and systemic anti-tumor immunity. Optically-active nanomedicine offers these features as well as spatiotemporal control of tumor-focused drug release to realize synergistic combination therapies. Achieving quantitative dosimetry is a major challenge, and dosimetry is fundamental to photomedicine for personalizing and tailoring therapeutic regimens to specific patients and anatomical locations. The challenge of dosimetry is perhaps greater for photomedicine than many standard therapies given the complexity of light delivery and light-tissue interactions as well as the resulting photochemistry responsible for tumor damage and drug-release, in addition to the usual intricacies of therapeutic agent delivery. An emerging multidisciplinary approach in oncology utilizes mathematical and computational models to iteratively and quantitively analyze complex dosimetry, and biological response parameters. These models are parameterized by preclinical and clinical observations and then tested against previously unseen data. Such calibrated and validated models can be deployed to simulate treatment doses, protocols, and combinations that have not yet been experimentally or clinically evaluated and can provide testable optimal treatment outcomes in a practical workflow. Here, we foresee the utility of these computational approaches to guide adaptive therapy, and how mathematical models might be further developed and integrated as a novel methodology to guide precision photomedicine.

Synergistic STING activation by PC7A nanovaccine and ionizing radiation improves cancer immunotherapy

Solid cancers are able to escape immune surveillance and are resistant to current treatment in immunotherapy. Recent evidence indicates the critical role of the stimulator of interferon genes (STING) pathway in antitumor immunity. STING-targeted activation is extensively investigated as a new strategy for cancer therapy. Previously, we reported a safe and efficacious STING-activating nanovaccine to boost systemic tumor-specific T cell responses in multiple tumor models. Local radiotherapy has been reported to not only reduce tumor burden but also enhance local antitumor immunity in a STING-dependent manner. In this study, we demonstrate that combination of these two modalities leads to a synergistic response with long-term regression of large established tumors in two mouse tumor models. The percentage of CD8+ T cells increased significantly in primary tumors after combination therapy. Mechanistically, the augmented T cell responses of radiotherapy and nanovaccine is STING pathway dependent. Furthermore, nanovaccine synergizes with radiotherapy to achieve a better therapeutic effect in distal tumors. These findings suggest that combination of local radiotherapy with systemic PC7A nanovaccine offers a useful strategy to improve the therapeutic outcome of late stage solid cancers.

PARP Inhibition Elicits STING-Dependent Antitumor Immunity in Brca1-Deficient Ovarian Cancer

SUMMARYPARP inhibitors have shown promising clinical activities for patients with BRCA mutations and are changing the landscape of ovarian cancer treatment. However, the therapeutic mechanisms of action for PARP inhibition in the interaction of tumors with the tumor microenvironment and the host immune system remain unclear. We find that PARP inhibition by olaparib triggers robust local and systemic antitumor immunity involving both adaptive and innate immune responses through a STING-dependent antitumor immune response in mice bearing Brca1-deficient ovarian tumors. This effect is further augmented when olaparib is combined with PD-1 blockade. Our findings thus provide a molecular mechanism underlying antitumor activity by PARP inhibition and lay a foundation to improve therapeutic outcome for cancer patients.

Cell-intrinsic and spatially divergent tumor programmed death ligand 1 (PD-L1) signals modify local and systemic anti-tumor immunity through novel chemokine effects

Abstract PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors > ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors > ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO> ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.

Liposomes-coated gold nanocages with antigens and adjuvants targeted delivery to dendritic cells for enhancing antitumor immune response

For nanovaccine-based cancer immunotherapy, dendritic cells (DCs) are one of the most powerful antigen presenting cells (APCs) that initiate and promote the maturation of antigen-specific cytotoxic T lymphocytes (e.g., CD8+ T cells) to induce the local and systemic antitumor immunity and further suppress the tumor metastasis and produce long-term protection against tumor. Thus, the activation and maturation of DCs is the prerequisite for efficient CD8+ T cell-based antitumor immune responses, which is considered as a primary and promising task for nanovaccine engineering. Herein, we introduce a versatile nanovaccine of liposomes-coated gold nanocages (Lipos-AuNCs) modified with DCs specific antibody aCD11c for targeted delivery of adjuvant MPLA and melanoma antigen peptide TRP2 to promote the activation and maturation of DCs, and enhance tumor specific T lymphocytes responses. Moreover, AuNCs accumulation and AuNCs-engulfed DCs migration to regional lymph nodes (RLNs) became real-time visualization through in vivo fluorescence and photoacoustic (PA) imaging to monitor the immunity process. In vivo experimental results demonstrated that the targeted antigen/adjuvants-loaded AuNCs exhibited enhanced antitumor immune response to inhibit tumor growth and metastasis in both B16-F10 prophylactic and lung metastasis models, which may act as a promising nanoplatform for antitumor immunotherapy and in vivo tracking.

Abstract B04: Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers

Abstract Background and Hypothesis: While the use of immune checkpoint blockade has gained significant traction as a viable therapy for certain cancers, many tumors remain refractory to these immunotherapies due to their highly immunosuppressive microenvironment. As a novel approach to boost local anti-tumor immunity and sensitize cancers to these therapies, we constructed an adenoviral (Ad) vector to overexpress the innate signaling adaptor molecule, Mitochondrial Antiviral Signaling protein (MAVS). Overexpression of MAVS results in innate immune signaling downstream of the RIG-I like receptors (RLRs) resulting in the activation of multiple signaling pathways as well as the release of a Th1 cytokine milieu and type I interferons. We therefore hypothesized that adenoviral delivery of MAVS into the tumor microenvironment would produce robust inflammatory signaling which would alter the tumor microenvironment to both efficiently suppress tumor growth and stimulate anti-tumor immune responses. Methods: In order to determine the possible effect MAVS would have on different cell types within the tumor microenvironment we infected a range of primary and cancer cells types (Bone-marrow derived dendritic cells (BMDCs), primary fibroblasts, as well as 5 different murine tumor cell lines representing breast-4T1, colon-CT26CL25, bladder-MB49, melanoma-B16F10, and prostate-TRAMPC2) with Ad-MAVS or a control Ad. Innate immune responses were measured via luminex ELISA, microarray, and RT-qPCR. The impact on tumor growth was assessed by MTT and expression of PD-L1 by FACS. Lastly, we measured the anti-tumor effect and adaptive immune responses of animals treated with various combinations of Ad-MAVS, a control Ad, and PD-L1 targeting antibodies in vivo utilizing various immunocompetent and immune-compromised mouse models. Results: In vitro infection of BMDCs, MEFs, and murine tumor cell lines demonstrated that MAVS expression elicited widespread production of cytokines and chemokines indicative of a type I immune response (such as IL-12, TNF-alpha, RANTES, etc.) and interferon beta. These profound responses also triggered immune regulatory feedback expression of PD-L1, enabling their targeting by PD-L1 antibodies. Additionally, we found that Ad-MAVS infection of tumor cell lines significantly inhibited their proliferation in vitro, likely via interferon pathways. In vivo, treatment with Ad-MAVS also elicited robust stimulation of innate immune responses and numerous type I inflammatory cytokines. Moreover, in multiple types of tumor bearing animals, we found that a single intralesional treatment of Ad-MAVS significantly prolonged overall survival and was capable of eliciting systemic anti-tumor adaptive immunity to non-immunogenic tumors. Conclusion: Our study identifies that MAVS expression can elicit the production of a significant number of Th1-type cytokines, chemokines, and interferon-beta in multiple cell types within the tumor microenvironment. We also demonstrate that overexpression of MAVS results in the direct suppression of tumor cell growth and stimulation of tumor PD-L1 expression. These activities were also observed in vivo, as intralesional injection of Ad-MAVS elicited Th1 cytokines and anti-tumor immunity in multiple tumor types, which correlated with enhanced systemic T-cell responses to tumor-specific epitopes. Collectively, these data demonstrate that intralesion injection of Ad-MAVS alters the tumor microenvironment to stimulate anti-tumor immunity and potentially sensitizes non-immunogenic tumors to PD-L1 therapies. As such, Ad-MAVS represents a novel approach of stimulating anti-tumor immunity via activation of innate adaptor molecules to improve immunogenic anti-tumor responses and potentially the therapeutic efficacy of PD-L1 immune checkpoint blockade. Citation Format: Gabriel De Leon, Tao Wang, Pankaj Agarwal, H. Kim Lyerly, Zachary Hartman. Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B04.

P3.02c-071 Spatially Selective Depletion of Regulatory T Cells with near Infrared Photoimmunotherapy for Syngeneic Lewis Lung Carcinoma: Topic: IT Biomarkers

CD4+CD25+Foxp3+ regulatory T-cells (Tregs) are known to suppress immune responses. Treg-mediated immunosuppression is a key mechanism for tumor immune-evasion, which could lead cancer immunotherapies to failures. Systemic depletion of Tregs has been tried; however, intravenously delivering antibodies against Tregs might not sufficiently deplete Tregs in tumor-microenvironment or could deplete effector cells. Moreover, auto-immune responses could cause potential side effects. To overcome these problems, we exploited near infrared photoimmunotherapy (NIR-PIT) at the tumor to deplete only intratumoral-Tregs. F(ab’)2 fragments of an anti-mouse CD25 antibody, PC61.3, were generated and conjugated with a phthalocyanine dye, IRDye-700DX (αnti-CD25-F(ab’)2-IR700). In vitro NIR-PIT effect was examined against CD25-expressing-T-lymphocytes, HT2-clone-A5E-cells. In vivo CD25-target-NIR-PIT was performed after an intravenous injection of the conjugate to mice bearing subcutaneous, luciferase transfected, LL/2 (Lewis lung carcinoma, LL/2-luc). Tumor-volume, bioluminescence signals (BLI), and Immune responses following the therapy were examined. In vitro NIR-PIT-induced cytotoxicity was light-dose-dependent. Local CD25-target-NIR-PIT selectively depleted intratumoral-Tregs; yet, Tregs in any other organs were not affected. The local CD25-target-NIR-PIT induced a intratumoral rapid activation and cytotoxic action of CD8-T cells and NK-cells. This led to significant reductions of tumor-volume (p < 0.0001) and BLI (p < 0.05) and prolonged survival (p < 0.0001) compared to non-treated controls. Intriguingly, this local CD25-target-NIR-PIT induced a transient systemic cytokine storm and antitumor-effects on distant non-irradiated specific tumors. Effects of local CD25-target-NIR-PIT were significantly (p < 0.0001) inhibited by a CD8-, NK-, or INFg-depletion, suggesting the anti-tumor roles of CD8 T-cells and NK-cells. Depletion of intratumoral-Tregs with a local CD25-target-NIR-PIT rapidly induced CD8 T- and NK-cell activation, thereby restoring local anti-tumor immunity. Consequently, activated immunity led to regression of not only NIR-PIT-treated-tumors but also non-NIR-light-exposed-tumors in separate parts of the body (Fig). These observations suggest that local CD25-target-NIR-PIT may be a promising new strategy for cancer immunotherapy.