B16F10 Tumor Research Articles

Abstract 2746: PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models

Abstract Cancer treatment has been revolutionized by targeting the interaction between tumors and immune system. For instance, PD-1/PD-L1 mono- or combo-therapy has achieved great success in clinics, and patient responsiveness is closed associated with PD-L1 expression in cancer cells, tumor infiltration leukocytes, and drug bio-distribution in the tumor microenvironment. We established a method to label PD-1 inhibitor with radioisotope 89Zr for in-life imaging (PET-CT). Anti-PD-1 sensitive mouse colorectal cell line MC38 and resistant mouse melanoma cell line B16F10 were implanted to C57BL/6 mice, which were then treated with 89Zr PD-1 inhibitor. PET imaging was performed at multiple time points to detect the bio-distribution of anti-PD-1, while the infiltrated immune cells were identified by FCM and PD-L1 expression measured by IHC. Notable tumor uptake of 89Zr PD-1 inhibitor was observed in mouse liver and tumor grafts. The uptake in tumor increased over time and plateaued 24 hours post-injection. MC38 and B16F10 tumor grafts showed difference in uptake levels. Blood and tumor at both flanks were removed for leucocyte phenotyping, and compared with B16F10, MC-38 tumors exhibited higher percentage of total lymphocytes and cytotoxic T lymphocytes, equivalent number of dendritic cells and less myeloid cells. Further phenotyping indicates that compared to MC-38, B16F10 tumors contain higher percentage of MDSC, most of which are monocytic myeloid derived suppressor cells. With our PET-CT/FACS platform, we can evaluate immunotherapy response and microenvironment changes in different tumors in “one-mouse” system. The unique immune microenvironment (high TIL, high CD8+ T and low MDSC) may contribute to the notable and sustainable uptake of PD-1 inhibitor and the corresponding efficacy. This system can act as a good translational platform to monitor the PK-PD readout of immune therapy. Citation Format: Xuzhen Tang, Fuyang Wang, Junyi Chen, Xuanming Zeng, Zhibo Liu, Qunsheng Ji. PET-CT/FACS platform to detect the biodistribution of radioisotope 89Zr PD-1 inhibitor in syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2746.

Abstract 2749: Age-related cancer drug toxicity and efficacy, and immune modulation in C57BL6J syngeneic model

Abstract Cancer has a higher incidence in the elderly and immunotherapy is widely applied to this population. However, the majority of preclinical studies assessing drug toxicity and efficacy have been done in young healthy mice, which could lead to translational failure. The goals of this study were to establish tumor models in aged mice for a better representation of cancer patients, and to examine the effects of aging on tumor growth, toxicity and efficacy of cancer treatment, and immune cell populations in syngeneic models using C57BL/6J mice of different ages. Young (7 weeks), mid-aged (56-58 weeks) and aged (76-78 weeks) C57BL/6J female mice were used for this study. To evaluate the maximum tolerated dose, young and aged mice were dosed with the standard of care cancer drugs (docetaxel, cisplatin, and doxorubicin) and body weight was monitored three times a week until mice reached endpoint criteria. Syngeneic tumor models were established using three different mouse cancer cell lines, B16-F10 melanoma, MC38 colon adenocarcinoma, and E0771 breast cancer. Several different concentrations of cells were injected subcutaneously and tumor growth kinetics were evaluated three times weekly. For efficacy study, mice were randomly assigned to isotype control or Anti-PD1 (8 mg/kg) treatment groups, and were dosed intravenously twice weekly for 3 weeks. One week after the last dose, whole blood and tumor samples were collected for flow cytometry analysis for immune cell profiling on B cells, T cells, NK cells. Aged mice showed higher toxicity against docetaxel, cisplatin, and doxorubicin compared to their young counterparts. Age-related tumor growth kinetics varied based on tumor models. Aging did not affect tumor growth kinetics of B16-F10 tumors, while aged mice showed slowed growth of MC38 tumors and faster growth of E0771 tumors. Across all tumor models, aged mice lost body weight while young mice gained body weight over the course of tumor growth, suggesting potential cancer-related cachexia in aged mice. Comparisons of PD-1 response in the various age groups with different tumors showed that anti-PD1 treatment significantly slowed down tumor growth in mid-aged (58 weeks) mice with B16-F10 tumors, but not in young or aged mice. However, the same treatment showed significant suppression of tumor growth in all age groups with the MC38 tumors. This effect might be partly caused by changes in the immune cell infiltration profile with aging. In conclusion, we have established syngeneic tumor models using aged mice and found aged mice exhibit higher toxicity against drugs while the effect of aging on tumor growth kinetics varies depending on models. Differences in immune cell infiltration in the tumor might be an underlying event that causes age-related differences in response to anti-PD1 treatment. The integration of aging in cancer research could potentially bridge the gap between preclinical studies and clinical outcomes. Citation Format: Jiwon Yang, Andrew Schile, Mingshan Cheng, James Keck. Age-related cancer drug toxicity and efficacy, and immune modulation in C57BL6J syngeneic model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2749.

Abstract 1477: LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy

Abstract In cancer blood vessels are dysfunctional, poorly perfused and leaky. Malfunctioning vessels contribute to the pro-oncogenic environment and limit the efficacy of current systemically administered drugs. Normalizing the tumor vasculature to improve vessel permeability, reduce hypoxia and vascular leakage and enhance drug delivery, has become an experimental objective in cancer research. This study was carried out to investigate the effect of blocking the secreted glycoprotein leucine-rich alpha-2-glycoprotein 1 (LRG1) on tumor vascular function, and evaluate the impact it has on the efficacy of the common standard of care chemotherapeutic drug cisplatin. Under normal conditions LRG1 is mainly expressed in the liver but also in other tissues such as bone marrow and immune cells. LRG1 has been described in multiple reports to be a serum prognostic biomarker in several cancers, for example lung, prostate, colorectal and breast. LRG1 promotes dysfunctional vessel growth by disrupting TGFβ signaling. We demonstrate that in Lrg1-/- mice and following treatment with a LRG1 function-blocking antibody (15C4) tumor growth was inhibited. In addition, we show using RNAscope that following subcutaneous grafting of the B16F0 and LL2 tumor cell lines in mice, Lrg1 is induced in tumor endothelial cells. Despite having no effect on total vessel area, the density was decreased upon LRG1 blockade, with the persisting larger vessels exhibiting improved vessel structure as evidenced by increased pericyte and basement membrane endothelial cell coverage. Better mural cell association with tumor vascular endothelial cells and basement membrane coverage are also indicators of vessel stabilization and maturation. Using a systemically delivered fluorescent lectin tracer to mark perfused vessels, we observed a significant increase in tumor perfusion in mice treated with 15C4. Lastly, vessel normalization, through LRG1 antibody blockade, significantly enhanced the efficacy of cisplatin chemotherapy as shown by a slower tumor growth rate and increased tumor cell death compared to monotherapy. These data further corroborate the hypothesis that inhibition of LRG1 improves the delivery, and hence efficacy, of a cytotoxic drug. In conclusion, deletion or inhibition of LRG1 results in an improved vascular configuration and function, and the efficacy of chemotherapy. LRG1 blockade may therefore represent a novel strategy to enhance vessel health and improve the efficacy of cancer therapeutics. Citation Format: Camilla Pilotti, Marie N O'Connor, David Kallenberg, Laura Dowsett, Jestin George, Stephen E. Moss, John Greenwood. LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1477.

Abstract 4585: LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth

Abstract The majority of Merkel cell carcinomas (MCC), a rare and highly-aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of a truncated form of the viral large T antigen (LT) in infected cells and making LT an attractive target for therapeutic cancer vaccines. While induction of tumor-reactive CD8+ T cells is a major goal of cancer therapy, CD4+ T cells provide essential support to CD8+ T cells by promoting their expression of cytotoxic effector molecules and increasing their migratory capacity. Cytokines secreted by CD4+ T cells, such as IFNγ, can also exert desirable effects on the tumor microenvironment. Therefore, we set out to design a cancer vaccine that promotes potent, antigen-specific CD4+ T cell responses to MCPyV-LT. To activate antigen-specific CD4+ T cells in vivo, we utilized our nucleic acid platform, UNITE (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and a balanced T cell response. LTS220A, encoding a mutated form of MCPyV-LT that abrogates its pro-oncogenic properties, was introduced into the UNITE platform. Intradermal vaccination with LTS220A-UNITE promoted a potent, antigen-specific CD4+ T cell response to MCPyV-LT. Additionally, prophylactic vaccination with LTS220A-UNITE prevented growth of B16F10 tumors expressing LTS220A in 100% of mice. Therefore, we find that DNA vaccination using the UNITE platform enhances CD4+ T cell responses to MCPyV-LT and prevents tumor growth when given prophylactically. Future studies will test the efficacy of LTS220A-UNITE for use as a therapeutic cancer vaccine. Citation Format: Claire Buchta Rosean, Pratima Sinha, David M. Koelle, Paul Nghiem, Teri Heiland. LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4585.

Abstract 2866: Neoantigen nanovaccine improves personalized cancer immunotherapy

Abstract Background: Neoantigens are attractive targets for personalized anti-tumor vaccination, given their uniqueness to the tumor and the ability to bypass immune tolerance. Recently, the feasibility of neoantigen vaccine has been demonstrated in patients. However, the response rate of neoantigen vaccines is unsatisfied because of their low immunogenicity, undesired degradation, limited cross-presentation, and acquired resistance. Here, we developed a nanoparticle-based neoantigen vaccine system to overcome these challenges. Methods: We predicted both MHC Class I and Class II neoantigen peptides for B16-F10 melanoma model using a bioinformatics pipeline. We screened an optimal incorporation strategy to formulate nanovaccines by either directly absorbing neoantigen peptides on PLGA nanoparticle (NP) or conjugating them to PEG-PLGA through a pH-responsive strategy or a redox-responsive strategy. We formulated nanovaccine with redox-responsive neoantigen-polymer conjugates and a STING agonist DMXAA. C57/BL6 mice were inoculated with 50,000 B16-F10 tumor cells on their right flank. Mice were vaccinated subcutaneously on their left flank with different nanovaccines or control arms on day 4, 8 and 12 after tumor inoculation. Mice were given anti-PD1 (200 μg, intraperitoneally) on day 4, 8, 12, 16, 20 after tumor inoculation. Tumor volume and mice survival were recorded. We also evaluated the immune related cytokines in mouse blood on day 15 after treatment. Results: Four MHC I and three MHC II neoantigen peptides were screened out for B16-F10 melanoma with high IFN-γ immune response. Results indicate that a redox conjugation strategy using PLGA-PEG and SPDP linker were more efficient in tumor inhibition than other incorporation strategies for our neoantigen peptides. By formulating nanovaccine with redox-responsive neoantigen-polymer conjugates and a STING agonist, we demonstrated that our nanovaccine, when combined with αPD1, achieved a 50% survival rate on day 38, compared to 0% of PBS treated group and 20% of non-formulated neoantigen peptides treated group. To confirm the enhanced immunity, we evaluated the immune related cytokines in mouse blood on day 15 after treatment. We found that our neoantigen nanovaccine achieved the highest expression of immune related cytokines among all arms, indicating that our nanovaccine induced higher immune response than non-formulated neoantigen peptides or other NP strategies. Conclusions: We demonstrate that our nanovaccine achieves increased therapeutic efficacy and higher expression of immune related cytokines than non-formulated neoantigen peptides. Our work develops a novel nanoparticle-based neoantigen vaccine that will improve current personalized cancer immunotherapy. Citation Format: Yu Mi, Christof C. Smith, Jonathan S. Serody, Benjamin G. Vincent, Andrew Z. Wang, Hyesun Hyun. Neoantigen nanovaccine improves personalized cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2866.

Conditional Deletion of PGC-1α Results in Energetic and Functional Defects in NK Cells.

SummaryDuring an immune response, natural killer (NK) cells activate specific metabolic pathways to meet the increased energetic and biosynthetic demands associated with effector functions. Here, we found in vivo activation of NK cells during Listeria monocytogenes infection-augmented transcription of genes encoding mitochondria-associated proteins in a manner dependent on the transcriptional coactivator PGC-1α. Using an Ncr1Cre-based conditional knockout mouse, we found that PGC-1α was crucial for optimal NK cell effector functions and bioenergetics, as the deletion of PGC-1α was associated with decreased cytotoxic potential and cytokine production along with altered ADP/ATP ratios. Lack of PGC-1α also significantly impaired the ability of NK cells to control B16F10 tumor growth in vivo, and subsequent gene expression analysis showed that PGC-1α mediates transcription required to maintain mitochondrial activity within the tumor microenvironment. Together, these data suggest that PGC-1α-dependent transcription of specific target genes is required for optimal NK cell function during the response to infection or tumor growth.

Abstract 4516: SA-4-1BBL as a novel agonist of CD137 has immunoprevention efficacy against various tumor types

Abstract Introduction: CD137 costimulation plays a paramount role in the generation of CD8+ T cell effector and long-term memory responses. Given the importance of CD8+ T cells for tumor destruction, we generated an oligomeric form of 4-1BB ligand, SA-4-1BBL, and demonstrated that it has better costimulatory activity and safety profile than an agonistic CD137 Ab. Importantly, prophylactic treatment of naïve mice with SA-4-1BBL as a single agent trains the immune system for long-term protection against cervical cancer. In this study, we expand on this initial observation and further demonstrate that the cancer immunoprevention efficacy of SA-4-1BBL extends to various tumor types and in different mouse strains, and that agonists Ab does not have such an attribute. Study design: Mice were treated subcutaneously with SA-4-1BBL protein or an agonistic Ab to CD137 receptor. Two weeks later, C57BL/6 mice were challenged subcutaneously in the left-back flank with syngeneic B16-F10 melanoma or 3LL lung cancer, whereas BALB/c mice were challenged with A20 lymphoma or 4T1 triple-negative breast cancer cell lines. Animals were monitor for tumor growth. For post-surgical tumor recurrences study, C57BL/6 mice with established B16-F10 tumors (~ 4mm in diameter) were subjected to surgery to debulk the tumor under sterile conditions. After 48 hours of the recovery period, animals were treated subcutaneously with SA-4-1BBL protein twice, two weeks apart. Animals without any treatment served as controls and mice were monitored for tumor relapse. Results: Pretreatment with SA-4-1BBL provided complete protection against both B16-F10 melanoma and 3LL lung cancer, ~80% protection against A20 lymphoma, and ~ 60% protection against triple negative 4T1 breast cancer. Importantly, the immunoprevention was a bona fide feature of SA-4-1BBL as the agonistic Ab to CD137 receptor had no impact on the tumor growth in the A20 lymphoma model tested. The immunoprotective effect was long lasting as challenge with the 3LL lung cancer cells 14 weeks post SA-4-1BBL treatment was still effective in controlling tumor growth in >80% of mice. SA-4-1BBL was also effective in controlling post-surgical recurrence against melanoma with ~75% efficacy. Ongoing RNA sequencing studies are aimed to dissect mechanistic basis of SA-4-1BBL-mediated cancer immunoprevention. Conclusions: These results demonstrate that SA-4-1BBL generates a blanket and long-lasting protective immune response against various tumor types. The ability of an immune checkpoint stimulator as a single agent to train the immune system for long-lasting protection independent of tumor type is exciting and sets the stage for a cancer immunoprevention modality that does not require tumor antigens. Funded in parts by NIH R41CA199956, T32HL134644, NCI R25CA134283 and Kentucky KSTC-184-512-16-237 awards. Citation Format: Haval Shirwan, Lalit Batra, Mohammad Tariq Malik, Pradeep Shrestha, Jenci L. Hawthorne, Esma S. Yolcu. SA-4-1BBL as a novel agonist of CD137 has immunoprevention efficacy against various tumor types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4516.

Abstract 6509: Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity

Abstract High doses of ionizing radiation (IR) as delivered with hypofractionated radiotherapy (HFRT) can generate immunogenic cell death and elicit adaptive anti-tumor immunity through cross priming of tumor associated antigens. Resiquimod (R-848), a TLR7/8 agonist, generates a systemic antitumor immunity resulting in tumor regression and enhanced T-cell effector function in cutaneous T-cell lymphomas clinically. We hypothesized that combining HFRT with immunotherapy would boost the antitumorigenic effects of either monotherapies, and if so, could lead to a readily translated clinical modality. B16F10 (murine melanoma) and SCC7 (murine squamous cell carcinoma) cells were used as to grow flank subcutaneous syngeneic tumor models in C57BL/6J and C3H/HeJ mice respectively. Our studies indicate that intratumoral administration of resiquimod combined with HFRT to those tumors leads to a significantly stronger antitumor response than either single treatment in both tumor models. The combination therapy in the primary tumor led to complete tumor regression of the SCC7 tumors in 86% of the treated mice to just 14% rejection in the HFRT group alone. Tumor re-challenge of those mice led to tumor rejection in 83% in the combination group compared to 0% of the HFRT group indicating a potent systemic effect of the combined therapy. Furthermore, the combination therapy led to significant reduction of the index (treated) but moreover in the abscopal (untreated) tumors compared to either monotherapy confirming a systemic antitumor effect in this dual-tumor in vivo model. RT-qPCR analysis revealed an increase expression of Th-1 signature (IFNγ, IL-12, Prf1, GzmB) in both primary and abscopal tumors and flowcytometric analysis of the abscopal B16F10 tumors revealed an increase in CD8+ infiltration. A more thorough flowcytometric analysis following staining with the gp100 tetramer revealed an increased cross-priming against gp100 (PMEL) and enhanced gp100-specific T-cell infiltration within the tumor. Moreover, splenocytes that were re-stimulated in vitro with the gp100 peptide in an IFNγ ELISPOT assay, they increased in mice treated with the dual therapy. Finally, an in vitro study of B16F10 cells, which express TLR7/8 receptors, revealed a substantial increase in IFNβ and CCL5 expression with the dual treatment. Knocking down TLR7 and Tmem173 (STING) in those cells with siRNAs completely reversed the phenotypic IFN type I expression signature. In conclusion, the combination of HFRT with Resiquimod leads to very potent antitumor effects in both primary and abscopal melanoma and squamous cell carcinoma tumors driven by an enhanced systemic immunologic response. Our findings provide the rational for translating therapies combining radiotherapy and intratumoral resiquimod into early-phase clinical trials. Citation Format: Ilias V. Karagounis, Stefano Pierini, Noëlle François, Christoforos Thomas, Xiaowei Xu, Constantinos Koumenis, Andrea Facciabene, Amit Maity. Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6509.

Abstract 4055: Immune checkpoint DNA vaccines enhance anti-tumor immunity in murine melanoma model

Abstract Background: Immune checkpoint blockade has emerged as an important cancer treatment strategy. However, the cost of repeat injections of these antibodies limits its availability to many patients globally. We have been developing DNA vaccines targeting immune checkpoints as a potentially more affordable approach to cancer immunotherapy. We have reported this platform could induce endogenous anti-CTLA-4 antibody and anti-tumor immunity. Here, we tested a DNA vaccine targeting PD-1 for anti-tumor responses. Methods: Murine CTLA-4 or PD-1 DNA sequences were inserted into an expression plasmid, pVAC-1, forming DNA vaccines targeting CTLA-4 or PD-1 respectively. Mouse melanoma models were established by inoculating B16F10 cells in the flank of C57BL/6 mice. The mice were randomized into treatment groups, including vector (control), radiation therapy (RT), or RT plus DNA vaccinations targeting CTLA-4 and/or PD-1. Vaccinations were given weekly via intra-muscular injection of the DNA plasmids in conjunction with electroporation. RT was given in 3 daily fractions of 8 Gy during the second week of vaccination. Serum samples were collected and subjected to ELISA for antibody titers. Tumors were measured twice weekly. Tumor samples were collected and subjected to IHC staining for immune markers. Results: Vaccination with plasmids encoding CTLA-4 (pVAC-1-CTLA-4) and PD-1 (pVAC-1-PD-1) induced endogenous antibodies against CTLA-4 and PD-1, respectively. The antibodies were detected beginning 2 weeks after the first vaccination, reached peak concentrations of 3-15 μg/mL in the following 1-2 weeks, and declined within a week once vaccination stopped. Compared with vector alone, preliminary studies using vaccination with both pVAC-1-CTLA-4 and pVAC-1-PD-1 suggested enhanced B16F10 tumor response without evidence of toxicities such as body weight loss or organ damage. In further preliminary studies, adding of both pVAC-1-CTLA-4 and pVAC-1-PD-1 DNA vaccines to RT resulted in increased CD8+ tumor infiltrating lymphocytes compared to either RT or vaccine alone and suggested enhanced tumor response. Conclusions: Immune checkpoint DNA vaccines induced endogenous specific antibodies, demonstrated a favorable safety profile, and may confer anti-tumor immunity. This DNA vaccine platform holds potential as a cost-effective approach to immunotherapy. Further studies are warranted to compare the effects of these immune checkpoint DNA vaccines with those of exogenous immune checkpoint targeting antibodies. Citation Format: Keng-Hsueh Lan, Raghava Sriramaneni, Justin C. Jagodinsky, Claire Baniel, Amy Erbe-Gurel, Jacquelyn A. Hank, Sung-Hsin Kuo, Keng-Li Lan, Zachary S. Morris. Immune checkpoint DNA vaccines enhance anti-tumor immunity in murine melanoma model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4055.

Abstract 4582: Tumor matrix-targeted polymeric glyco-adjuvant for in situ cancer vaccination

Abstract Cancer vaccination is challenging due to a lack of strong, clinically approved adjuvants and difficulty in identifying cancer-specific antigens. Here, in order to address these challenges, we report the creation of a targeted therapeutic cancer vaccine with two components: (1) a mannosylated, toll-like receptor 7 (TLR7)-agonizing polymer, p(Man-TLR7), and (2) a fusion protein consisting of serum albumin (SA) fused to a collagen binding domain (CBD), namely the A3 domain of von Willebrand factor. p(Man-TLR7) has been shown to elicit strong CD8+ T cell responses, which are vital for the success of cancer vaccines. As such, we hypothesized that chemically linking p(Man-TLR7) to a tumor-targeting protein would localize our polymeric glyco-adjuvant to the tumor, promoting the immunogenic processing of endogenous tumor antigens and bypassing the need to pre-identify tumor-specific antigens. In the approach presented here, CBD-SA acts as our tumor-targeting protein by actively binding to collagen in the tumor microenvironment via the CBD, in addition to utilizing the passive targeting of SA. Upon successful and reproducible generation of CBD-SA-p(Man-TLR7) conjugates, we observed that the conjugates retained the ability to bind both collagen I and collagen III. We then assessed the in vivo anti-tumor efficacy of our conjugates in B16F10 tumor-bearing mice, testing both murine CBD-SA-p(Man-TLR7) and human CBD-SA-p(Man-TLR7) conjugates to evaluate the translatability of our approach. We observed an 84% decrease in average B16F10 tumor size and improved overall survival compared to untreated controls when mice were treated with intravenously-delivered murine CBD-SA-p(Man-TLR7) in combination with anti-PD-1 and anti-CTLA-4 antibodies. In the case of intravenously-delivered human CBD-SA-p(Man-TLR7), a 60% decrease in average tumor size compared to untreated controls was observed when mice were treated with our vaccine as a monotherapy. Further improved anti-tumor efficacy (an 85% decrease in average tumor size compared to untreated controls) and improved overall survival were observed when we combined our human CBD-SA-p(Man-TLR7) vaccination with anti-PD-1 and anti-CTLA-4 antibodies. In conclusion, in situ vaccination with CBD-SA-p(Man-TLR7) is a translatable approach that synergizes with checkpoint antibody therapy to provide therapeutic benefit in treatment of a poorly immunogenic melanoma model. Citation Format: Laura T. Gray, D. Scott Wilson, Jun Ishihara, Koichi Sasaki, Michal Raczy, Melody Swartz, Jeffrey Hubbell. Tumor matrix-targeted polymeric glyco-adjuvant for in situ cancer vaccination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4582.

Discovery of Novel Resorcinol Dibenzyl Ethers Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction as Potential Anticancer Agents.

Novel small molecule compounds based on various scaffolds including chalcone, flavonoid, and resorcinol dibenzyl ether were designed and tested for their inhibitory activity against the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 (PD-1/PD-L1) pathway. Among them, compound NP19 inhibited the human PD-1/PD-L1 interaction with IC50 values of 12.5 nM in homogeneous time-resolved fluorescence (HTRF) binding assays. In addition, NP19 dose-dependently elevated IFN-γ production in a coculture model of Hep3B/OS-8/hPD-L1 and CD3 T cells. Furthermore, NP19 displayed significant in vivo antitumor efficacy in two different mouse models of cancer (a melanoma B16-F10 tumor model and an H22 hepatoma tumor model). Moreover, H&E staining and flow cytometry data suggested that NP19 activated the immune microenvironment in the tumor, which may contribute to its antitumor effects. This work shows NP19 is a promising lead compound for further development as a new generation of small molecule inhibitors targeting the PD-1/PD-L1 pathway.

Nanovaccine’s rapid induction of anti-tumor immunity significantly improves malignant cancer immunotherapy

• IV priming and SC boosting vaccination strategy most efficiently inhibits malignant tumor growth. • Delivering nanovaccines to spleen promotes rapid and strong Th1-biased anti-tumor immunity. • Nanovaccine depot formed after SC injection slowly induces potent and durable anti-tumor immunity. • Simultaneous IV/SC vaccination strategy significantly delays the progress of advanced tumors. The advantage of rapid tumor growth over slow immune induction often limits the efficiency of cancer therapeutic vaccines. Many functionalized micro-/nano-vaccines are shown to enhance cancer immunotherapy, but few studies correlate the therapeutic efficacy of these micro-/nano-vaccines to the vaccination route such as intravenous (IV), subcutaneous (SC) injection or their combination. Herein, we employed classical “priming + boosting” vaccination strategy to investigate the influence of four vaccination combinations (IV + IV, IV + SC, SC + IV and SC + SC) of nanovaccines on the anti-tumor therapeutic efficacy in mice bearing E.G7-OVA-lymphoma and B16F10-melanoma. The nanovaccines were constructed by loading antigen and CpG onto layered double hydroxide (LDH) nanoparticles. Our experimental data indicate that “IV-priming + SC-boosting” vaccination combination most efficiently inhibits the growth of early stage tumors, with the tumor volume reduced by >75-90 % in comparison with the control group. Based on these findings, a novel vaccination strategy, i.e. simultaneous IV and SC injection was proposed, which significantly delayed the progression of both early stage and advanced B16F10 tumors. Our current research for the first time correlates the vaccination route of nanovaccines to the anti-tumor therapeutic efficacy, and the highest efficacy of optimal vaccination combination (IV + SC) is benefited from that fact that IV priming quickly induces strong anti-tumor responses that are well sustained by subsequent SC boosting. Moreover, the simultaneous IV/SC vaccination potentially provides a novel vaccination strategy for enhanced immunotherapy of early stage and advanced tumors.

Tumor cell membrane enveloped aluminum phosphate nanoparticles for enhanced cancer vaccination

An ideal cancer vaccine should contain both strongly immunogenic cancer-specific antigen and potent adjuvant for stimulating robust cellular immunity which are pivotal for clearance of cancer cells. However, most of commercially available adjuvants such as aluminum phosphate gel cannot stimulate robust cellular immune response. In the current study, we reformed microscale aluminum phosphate gel adjuvant into nanoscale and fabricated CpG loaded and B16F10 tumor cell membrane coated aluminum phosphate nanoparticles (APMC). The resultant nano-vaccines showed a size of around 60 nm and a negative surface charge of −40 mV. Tumor cell membrane not only served as tumor antigens but also effectively improved the colloidal dispersion of aluminum phosphate nanoparticles. Subcutaneously injected APMC were efficiently drained to mouse lymph nodes, significantly increased co-uptake of tumor antigen and CpG by lymph node resident antigen presenting cells, promoted maturation of these cells and enhanced lysosomal antigen escape. After immunizing mice, they triggered robust cellular immunity, including potent IFN-γ+CD4+ T cells, IFN-γ+CD8+ T cells, cytotoxic T lymphocytes and cytokine excretion in spleen and lymph node cells. The elicited responses significantly suppressed tumor growth and prolonged survival of mice in both prophylactic and therapeutic melanoma models. This promising vaccine delivery system shows great potential to clinical transformation and can be further developed for personalized cancer vaccines.

Exosome-Containing Preparations From Postirradiated Mouse Melanoma Cells Delay Melanoma Growth In Vivo by a Natural Killer Cell–Dependent Mechanism

To investigate the ability of radiation to stimulate exosome release from melanoma cells and to characterize the resulting exosome-containing vesicle preparations for their ability to promote a host antitumor immune response. Cultured B16F10 murine melanoma cells or tumors were irradiated, and secreted extracellular vesicles were isolated and characterized. The exosome-containing vesicle preparations were injected into fresh tumors in syngeneic mice, and tumor growth and infiltrating T cells and natural killer (NK) cells were characterized. Irradiation stimulated exosome release from B16F10 murine melanoma cells. Exosome preparations from irradiated cell culture supernatants were biologically active, as demonstrated by uptake into recipient cells and by the ability to induce dendritic cell maturation and activation in vitro. Intratumoral injection significantly delayed tumor growth in vivo, whereas injection of similar preparations from non irradiated cells had no effect. The antitumor effect was correlated to an increase in interferon gamma-producing tumor-infiltrating NK cells. Pretreatment of the host mice with anti-NK cell antibodies abolished the effect, whereas pretreatment with anti-CD8+ T-cell antibodies did not. Exosomes from irradiated cells, or synthetic mimics, might provide an effective strategy for potentiation of NK cell-mediated host antitumor immunity.

The pore size of mesoporous silica nanoparticles regulates their antigen delivery efficiency.

Subunit vaccines generally proceed through a 4-step in vivo cascade-the DUMP cascade-to generate potent cell-mediated immune responses: (1) drainage to lymph nodes; (2) uptake by dendritic cells (DCs); (3) maturation of DCs; and (4) Presentation of peptide-MHC I complexes to CD8+ T cells. How the physical properties of vaccine carriers such as mesoporous silica nanoparticles (MSNs) influence this cascade is unclear. We fabricated 80-nm MSNs with different pore sizes (7.8 nm, 10.3 nm, and 12.9 nm) and loaded them with ovalbumin antigen. Results demonstrated these MSNs with different pore sizes were equally effective in the first three steps of the DUMP cascade, but those with larger pores showed higher cross-presentation efficiency (step 4). Consistently, large-pore MSNs loaded with B16F10 tumor antigens yielded the strongest antitumor effects. These results demonstrate the promise of our lymph node-targeting large-pore MSNs as vaccine-delivery vehicles for immune activation and cancer vaccination.

The Effect of Phase Transition Temperature on Therapeutic Efficacy of Liposomal Bortezomib.

Here, three liposomal formulations of DPPC/DPPG/Chol/DSPE-mPEG2000 (F1), DPPC/DPPG/Chol (F2) and HSPC/DPPG/Chol/DSPE-mPEG2000 (F3) encapsulating BTZ were prepared and characterized in terms of their size, surface charge, drug loading, and release profile. Mannitol was used as a trapping agent to entrap the BTZ inside the liposomal core. The cytotoxicity and anti-tumor activity of formulations were investigated in vitro and in vivo in mice bearing tumor. Bortezomib (BTZ) is an FDA approved proteasome inhibitor for the treatment of mantle cell lymphoma and multiple myeloma. The low solubility of BTZ has been responsible for the several side effects and low therapeutic efficacy of the drug. Encapsulating BTZ in a nano drug delivery system; helps overcome such issues. Among NDDSs, liposomes are promising diagnostic and therapeutic delivery vehicles in cancer treatment. Evaluating anti-tumor activity of bortezomib liposomal formulations. Data prompted us to design and develop three different liposomal formulations of BTZ based on Tm parameter, which determines liposomal stiffness. DPPC (Tm 41°C) and HSPC (Tm 55°C) lipids were chosen as variables associated with liposome rigidity. In vitro cytotoxicity assay was then carried out for the three designed liposomal formulations on C26 and B16F0, which are the colon and melanoma cancer mouse-cell lines, respectively. NIH 3T3 mouse embryonic fibroblast cell line was also used as a normal cell line. The therapeutic efficacy of these formulations was further assessed in mice tumor models. MBTZ were successfully encapsulated into all the three liposomal formulations with a high entrapment efficacy of 60, 64, and 84% for F1, F2, and F3, respectively. The findings showed that liposomes mean particle diameter ranged from 103.4 to 146.8nm. In vitro cytotoxicity studies showed that liposomal-BTZ formulations had higher IC50 value in comparison to free BTZ. F2-liposomes with DPPC, having lower Tm of 41°C, showed much higher anti-tumor efficacy in mice models of C26 and B16F0 tumors compared to F3-HSPC liposomes with a Tm of 55°C. F2 formulation also enhanced mice survival compared with untreated groups, either in BALB/c or in C57BL/6 mice. Our findings indicated that F2-DPPC-liposomal formulations prepared with Tm close to body temperature seem to be effective in reducing the side effects and increasing the therapeutic efficacy of BTZ and merits further investigation.

MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA.

NKG2D is a potent activating immunoreceptor expressed on nearly all cytotoxic lymphocytes promoting their cytotoxicity against self-cells expressing NKG2D ligands (NKG2DLs). NKG2DLs are MHC class I-like glycoproteins that usually are not expressed on “healthy” cells. Rather, their surface expression is induced by various forms of cellular stress, viral infection, or malignant transformation. Hence, cell surface NKG2DLs mark “dangerous” cells for elimination by cytotoxic lymphocytes and therefore can be considered as “kill-me” signals. In addition, NKG2DLs are up-regulated on activated leukocytes, which facilitates containment of immune responses. While the NKG2D receptor is conserved among mammals, NKG2DL genes have rapidly diversified during mammalian speciation, likely due to strong selective pressures exerted by species-specific pathogens. Consequently, NKG2DL genes are not conserved in man and mice, although their NKG2D-binding domains maintained structural homology. Human NKG2DLs comprise two members of the MIC (MICA/MICB) and six members of the ULBP family of glycoproteins (ULBP1–6) with MICA representing the best-studied human NKG2DLs by far. Many of these studies implicate a role of MICA in various malignant, infectious, or autoimmune diseases. However, conclusions from these studies often were limited in default of supporting in vivo experiments. Here, we report a MICA transgenic (MICAgen) mouse model that replicates central features of human MICA expression and function and, therefore, constitutes a novel tool to critically assess and extend conclusions from previous in vitro studies on MICA. Similarly to humans, MICA transcripts are broadly present in organs of MICAgen mice, while MICA glycoproteins are barely detectable. Upon activation, hematopoietic cells up-regulate and proteolytically shed surface MICA. Shed soluble MICA (sMICA) is also present in plasma of MICAgen mice but affects neither surface NKG2D expression of circulating NK cells nor their functional recognition of MICA-expressing tumor cells. Accordingly, MICAgen mice also show a delayed growth of MICA-expressing B16F10 tumors, not accompanied by an emergence of MICA-specific antibodies. Such immunotolerance for the xenoantigen MICA ideally suits MICAgen mice for anti-MICA-based immunotherapies. Altogether, MICAgen mice represent a valuable model to study regulation, function, disease relevance, and therapeutic targeting of MICA in vivo.

Immuno-SPECT/PET imaging with radioiodinated anti-PD-L1 antibody to evaluate PD-L1 expression in immune-competent murine models and PDX model of lung adenocarcinoma

ObjectiveAccurate evaluation of tumor programmed death ligand 1 (PD-L1) expression can assist in predicting whether a patient will respond to anti-PD-L1 therapy. In this study, we aimed to develop stable radioiodinated PD-L1 antibodies that can be used for PD-L1 targeted SPECT/PET imaging. MethodsRadioiodination was accomplished via a prosthetic group ([131I]SIB or [124I]SIB) to give radioiodinated anti-human PD-L1 and anti-mouse PD-L1 antibody (anti-PD-L1 and anti-PD-L1M). MicroSPECT/PET imaging and biodistribution of radioiodinated antibodies were studied in two immune-competent murine models (B16F10 and 4T1 syngeneic tumor models) and patient-derived xenograft (PDX) model of lung adenocarcinoma to evaluate the feasibility of identifying tumor PD-L1 expression. ResultsRadioiodinated PD-L1 antibodies had high radiochemical purity (>99%) and favorable stability in vivo. There was high uptake of [131I]SIB-anti-PD-L1M in both 4T1 and B16F10 syngeneic tumors when injected with 5.5 MBq radiotracers containing 200 μg anti-mouse-PD-L1. The presence of excess unlabeled anti-PD-L1 antibody increased [131I]SIB-anti-PD-L1M uptake in tumors. The highly specific PD-L1-positive tumor uptake detected by SPECT imaging indicated that radioiodinated antibody could be used for PD-L1 expression imaging. In addition, PET imaging of the PDX model was performed with [124I]SIB-anti-PD-L1, which showed high signal intensity in tumors and optimal contrast between tumor and muscle (tumor-to-muscle ratios at 6 h p.i. and 24 h p.i. were 2.5 and 5.3, respectively). ConclusionsThis study provides an efficient strategy for synthesizing stable radioiodinated PD-L1 antibodies with excellent pharmacokinetics to identify PD-L1 expression in tumors.

Effective pressure and treatment duration of high hydrostatic pressure to prepare melanoma vaccines.

Current therapeutic methods for melanoma have numerous limitations, and thus the improvement of such treatment methods are essential. One possible option is the vaccination of autologous inactivated tumor cells. The primary indispensable principles of a cell-based melanoma vaccine include: i) Entire inactivation of melanoma cells; ii) retaining the immunogenicity of melanoma cells; and iii) adherence to laws and ethical guidelines. However, traditional methods for the production of the vaccine, such as ultrasonic, chemotherapeutics and freeze-thawing, have some juridical or therapeutic constraints. Therefore, the present study used high hydrostatic pressure (HHP) to inactivate malignant cells, and treated B16-F10 tumor cells with different pressures (≥50 MPa) and different durations (≥1 min). It was identified that tumor cells in vitro lost their proliferative ability, but retained their immunogenicity following treatment. Furthermore, the vaccination of the melanoma cells significantly suppressed their oncogenesis. Collectively, the present results suggest that HHP treatment may be an economically viable and effective measure to develop a melanoma vaccine, when pressure was ≥200 MPa and the treatment duration was ≥30 min.

Combination immunotherapy of oncolytic virus nanovesicles and PD-1 blockade effectively enhances therapeutic effects and boosts antitumour immune response

Immunotherapies are changing the landscape of melanoma treatment, but 70% of the melanoma patients have no response to immune checkpoint inhibitors or oncolytic virus therapy. Thus, novel formulations are needed to improve the population benefiting from immunotherapy. Here, we report a combined therapeutic modality based on oncolytic virus nanovesicles composed of CaCl2, oncolytic virus Ad5, lecithin and cholesterol (Lipo-Cap–Ad5) with immune checkpoint blockade (anti-PD-1 antibody). We investigated in vivo antitumour activity, systemic toxicity and mechanism of antitumour immune responses of Lipo-Cap–Ad5 + anti-PD-1 blockade, in a murine B16F10 tumour xenograft model. Through a series of in vivo studies, we found that Lipo-Cap–Ad5 in combination with anti-PD-1 blockade drastically reduced the tumour growth by 76.6%, and prolonged animals’ survival with no obvious toxicity observed in heart, liver and kidney. The combination therapy facilitates tumour infiltration of effector CD4+, CD8+ T cells and increases secretion of TNF-α and IFN-γ. Therefore, Lipo-Cap–Ad5 in combination with anti-PD-1 blockade can potentiate and activate the immune system synergistically, ultimately creating a pro-inflammatory environment. These results suggest that combination immunotherapy of Lipo-Cap–Ad5 and anti-PD-1 blockade developed in this study has promising applications to enhance therapeutic efficacy with the potential of being translated into clinical practice.