Tumor Cell Rechallenge Research Articles

Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity.

mRNA neoantigen cancer vaccine inducing neoantigen-specific T cell responses holds great promise for cancer immunotherapy; however, its clinical translation remains challenging because of suboptimal neoantigen prediction accuracy and low delivery efficiency, which compromise the in vivo therapeutic efficacy. We present a lipopolyplex (LPP)-formulated mRNA cancer vaccine encoding tandem neoantigens as a cancer therapeutic regimen. The LPP-formulated mRNA vaccines elicited robust neoantigen-specific CD8+ T cell responses in three syngeneic murine tumor models (CT26, MC38, and B16F10) to suppress tumor growth. Prophylactic cancer vaccine treatment completely prevented tumor development, and long-lasting memory T cells protected mice from tumor cell rechallenge. Combining the vaccine with immune checkpoint inhibitor further boosted the antitumor activity. Of note, LPP-based personalized cancer vaccine was administered in two cancer patients and induced meaningful neoantigen-specific T cell and clinical responses. In conclusion, we demonstrated that the LPP-based mRNA vaccine can elicit strong antitumor immune responses, and the results support further clinical evaluation of the therapeutic mRNA cancer vaccine.

A novel vesicular stomatitis virus armed with IL-2 mimic for oncolytic therapy

Oncolytic virus (OV) is increasingly being recognized as a novel vector in cancer immunotherapy. Increasing evidence suggests that OV has the ability to change the immune status of tumor microenvironment, so called transformation of ‘cold’ tumors into ‘hot’ tumors. The improved anti-tumor immunity can be induced by OV and further enhanced through the combination of various immunomodulators. The Neo-2/15 is a newly de novo synthesized cytokine that functions as both IL-2 and IL-15. However, it specifically lacks the binding site of IL-2 receptor α subunit (CD25), therefore unable to induce the Treg proliferation. In present study, a recombinant vesicular stomatitis virus expressing the Neo-2/15 (VSVM51R-Neo-2/15) was generated. Intratumoral delivery of VSVM51R-Neo-2/15 efficiently inhibited tumor growth in mice without causing the IL-2-related toxicity previously observed in clinic. Moreover, treatment with VSVM51R-Neo-2/15 increased the number of activated CD8+ T cells but not Treg cells in tumors. More tumor-bearing mice were survival with VSVM51R-Neo-2/15 treatment, and the surviving mice displayed enhanced protection against tumor cell rechallenge due to the induced anti-tumor immunity. In addition, combination therapy of OV and anti-PD-L1 immune checkpoint inhibitors further enhanced the anti-tumor immune response. These findings suggest that our novel VSVM51R-Neo-2/15 can effectively inhibit the tumor growth and enhance the sensitivity to immune checkpoint inhibitors, providing promising attempts for further clinical trials.

Abstract LB074: Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies

Abstract Autologous chimeric antigen receptor (CAR)-T therapies have elicited high rates of durable complete responses in B cell lymphomas. Unfortunately, disease progression, manufacturing failures, and prohibitive costs highlight the need for an alternative source of cells that would provide a consistent and readily available therapy for patients. The “Appia Cells Utilized for Allogeneic” (ACUA) platform is an innovative process that differentiates cord blood-derived CD34+ hematopoietic stem and progenitor cells into large numbers of natural killer T (NKT) cells to support off-the-shelf treatment. NKT cells express an invariant T cell receptor (iTCR) and are naturally non-alloreactive, making them an ideal cell type for an allogeneic product. The ACUA platform circumvents the challenge of expanding NKT cells from the periphery where the cells comprise less than 1% of T cells. API-192 is an ACUA-derived, NKT cell product engineered to express dual CARs targeting CD19 and CD20 to prevent tumor antigen escape. At research scale, API-192 is a highly pure product (>90% iTCR+ 19CAR+ 20CAR+) that secretes soluble interleukin-15 for improved expansion and persistence. The transgenic iTCR is not expressed above physiologic levels and upon engagement, API-192 proliferates in response to and is cytotoxic against CD1d+ tumor cells in a ligand-dependent manner. In response to Raji and Nalm6 tumor cells, API-192 secretes pro-inflammatory cytokines, proliferates, and is cytotoxic in a dose- and CAR-dependent manner. API-192 can also control multiple rounds of tumor cell rechallenge in vitro. Using human tumor xenograft mouse models, we show that API-192 expands, controls tumor growth, and prolongs survival. Furthermore, API-192 persists in the periphery, enabling control of a tumor rechallenge at 70 days after initial dosing of effector cells. Indicative of a favorable immunogenic profile, API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less interferon (IFN)-γ release by NK and T cells in a mixed lymphocyte reaction (MLR) assay. To address a theoretical concern that endogenous TCRαβ rearrangement could occur during the ACUA process, we performed genomic DNA based TCR sequencing to quantify the level of TCRα or TCRβ rearrangement and preliminary data suggested negligible levels (<0.2%). Using a MLR assay, we demonstrate that compared to conventional CAR-T cells, API-192 secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with a low graft vs host risk. In an autonomous growth assay, API-192 expands at a low level and subsequently contracts, suggesting a lack of dysregulated growth. Collectively, these results demonstrate the favorable manufacturability, potency, and safety of API-192 and its potential as a novel allogeneic therapy. Citation Format: Lanny Gov, Jiaji Yu, Maha Qubain, Saige L. Pompura, Sean D. Allen, Michael A. Christopher, Jeff Wiezorek. Preclinical development of API-192: An engineered CAR-NKT allogeneic cell therapy targeting CD19 and CD20 for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB074.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

Low-dose total body irradiation enhances systemic anti-tumor immunity induced by local cryotherapy.

Strategies that restore the immune system's ability to recognize malignant cells have yielded clinical benefits but only in some patients. Tumor cells survive cryotherapy and produce a vast amount of antigens to trigger innate and adaptive responses. However, because tumor cells have developed immune escape mechanisms, cryotherapy alone may not be enough to induce a significant immune response. The mice were randomly divided into four groups: Group A: low-dose total body irradiation combined with cryotherapy (L-TBI+cryo); Group B: cryotherapy (cryo); Group C: low-dose total body irradiation(L-TBI); Group D: control group (Control). The tumor growth, recurrence, and survival time of mice in each group were compared and the effects of different treatments on systemic anti-tumor immunity were explored. L-TBI in conjunction with cryotherapy can effectively control tumor regrowth, inhibit tumor lung metastasis, extend the survival time of mice, and stimulate a long-term protective anti-tumor immune response to resist the re-challenge of tumor cells. The anti-tumor mechanism of this combination therapy may be related to the stimulation of inflammatory factors IFN-γ and IL-2, as well as an increase in immune effector cells (CD8+ T cells) and a decrease in immunosuppressive cells (MDSC, Treg cells) in the spleen or tumor tissue. We present unique treatment options for enhancing the immune response caused by cryotherapy, pointing to the way forward for cancer treatment.

Abstract 6225: Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models

Abstract Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios <= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6225.

Nitric Oxide Induces Immunogenic Cell Death and Potentiates Cancer Immunotherapy.

Tumor cells undergoing immunogenic cell death (ICD) release immunogenic damage-associated molecular patterns (DAMPs) to trigger a long-term protective antitumor response. ICD can be induced by certain pathogens, chemotherapeutics, and physical modalities. In this work, we demonstrate that a gaseous molecule, specifically nitric oxide (NO), can induce a potent ICD effect. NO exerts cytotoxic effects that are accompanied by the emission of DAMPs based on the endoplasmic reticulum stress and mitochondrial dysfunction pathways. Released DAMPs elicit immunological protection against a subsequent rechallenge of syngeneic tumor cells in immunocompetent mice. We prepare polynitrosated polyesters with high NO storage capacity through a facile polycondensation reaction followed by a postsynthetic modification. The polynitrosated polyesters-based NO nanogenerator (NanoNO) that enables efficient NO delivery and controlled NO release in tumors induces a sufficient ICD effect. In different immune-intact models of tumors, the NanoNO exhibits significant tumor growth suppression and increases the local dose of immunogenic signals and T cell infiltrations, ultimately prolonging survival. In addition, the NanoNO synergizes with the PD-1 blockade to prevent metastasis. We conclude not only that NO is a potent ICD inducer for cancer immunotherapy but also that it expands the range of ICD inducers into the field of gaseous molecules.

Abstract 1792: Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model

Abstract Background: Tumor-associated macrophages (TAMs) promote tumor progression and resistance to immune checkpoint inhibitors and are thus attractive targets for cancer immunotherapy. The STAT6 transcriptional network is an important driver of the immune-suppressive M2 macrophage program in the tumor microenvironment (TME). Previous attempts to therapeutically target these transcriptional networks have not been successful. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. Leveraging the potential of exosomes, we have developed a novel, engineered exosome therapeutic candidate loaded with antisense oligonucleotides (ASO) targeting STAT6 (exoASO-STAT6), that effectively silences STAT6 expression in TAMs. Results: In vitro and in vivo studies demonstrate an enhanced delivery of ASO to M2 macrophages. exoASO showed a 2x improvement in uptake vs free ASO in human M2 macrophages in vitro. Following IV administration, exoASO demonstrated up to 11x increase in uptake in monocytes and MDSCs in the blood, Kupffer cells in liver and TAMs and MDSCs in the tumor. In vitro treatment with exoASO-STAT6 resulted in 90% target gene KD in human, mouse and cynomolgus monkey M2 macrophages, which was persistent for up to 10 days. Additionally, exoASO-STAT6 demonstrated greater potency than free ASO. STAT6 KD resulted in a 7x decrease in M2 marker CD163 and a 25x increase in pro-inflammatory cytokines such as IL-12 or TNFα, demonstrating effective macrophage reprogramming. In vivo efficacy studies in CT26 showed potent dose-dependent single agent activity of exoASO-STAT6, with a cumulative dose of 36 μg of ASO resulting in 94% TGI and 80% complete responses. CD8 T-cell depletion abrogated anti-tumor activity and the complete responders were resistant to tumor cell re-challenge, demonstrating a CD8-T cell mediated immunological memory response. In an orthotopic model of HCC that is resistant to anti-PD-1 or anti-CSF1R therapy, IV administration of exoASO-STAT6 significantly attenuated tumor growth, as observed by a 61% reduction in tumor mass and complete elimination of tumor lesions in 50% of treated mice. exoASO-STAT6 therapy resulted in a decrease in M2 markers such as Tgfb1 and Ccl17 and an increase in M1 markers such as IL1b. A significant increase in interferon and cytotoxic T-cell gene signatures was also observed, demonstrating effective reprogramming of the TME. Conclusion: exoASO-STAT6 is a novel therapeutic that selectively targets STAT6, a key transcription factor in TAMs. This therapy results in effective macrophage reprogramming to a pro-inflammatory M1 phenotype and potent single agent anti-tumor activity in multiple checkpoint refractory tumor models. In sum, exoASO-STAT6 represents a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, Su Chul Jang, Christine McCoy, Kelvin Zhang, William Dahlberg, Marie Leblanc, Hugo Quillery, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1792.

Reducing the immunosuppressive tumor microenvironment enhances photoimmunotherapy efficacy.

Reducing the immunosuppressive tumor microenvironment enhances photoimmunotherapy efficacy.

Mannose-Derived Carbon Dots Amplify Microwave Ablation-Induced Antitumor Immune Responses by Capturing and Transferring “Danger Signals” to Dendritic Cells

Hepatocellular carcinoma recurrence and metastasis after microwave ablation (MWA) are challenges in the clinic. This study showed that mannose-derived carbon dots (Man-CDs) could effectively capture several "danger signals" (DS) after MWA treatment and then deliver DS specifically to dendritic cells (DCs). This improved delivery of DS to DCs enhanced the processing and presentation of tumor-associated antigens by DCs. The results demonstrated that intratumoral injection of Man-CDs after MWA therapy elicited a potent tumor-specific immune response and finally led to the effective suppression of both primary and distant tumors. MWA + Man-CD treatment could efficiently reject tumor cell rechallenge in vivo. This study demonstrated that Man-CD nanoparticles are effective adjuvants that can improve MWA therapy by eliciting a tumor-specific immune response.

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas. We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression. We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models. Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.

Abstract 6686: SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner

Abstract SO-C101 (RLI-15) is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells and reducing the toxicity of IL-15 as such. SO-C101 was previously shown to exhibit a potent anti-metastatic activity in Renca, B16F10 melanoma and delayed tumor growth in T cell-based mouse tumor models (CT26, MC38). Here we investigated the anti-tumor efficacy in predominantly natural killer (NK)-cell based mouse tumor models TC-1 and TRAMP-C2. We showed that SO-C101 monotherapy was effective in the treatment of established TC-1 tumors, which was dependent on the presence of both NK and CD8+ T cells, but not CD4+ T cells. In an early treatment setting SO-C101 significantly decreased the rate of tumor development also in dependence on NK and CD8+ T cells. SO-C101 effectively reduced tumor growth in TRAMP-C2 mice in early and advanced treatment settings. However, only in combination with anti-PD-1 antibody treatment the tumor development was prevented in majority of mice. This effect was durable, and the new tumor development was further significantly delayed after a tumor cell re-challenge, which suggests the involvement of memory T cells despite an important NK cell role in anti-tumor efficacy in these models. The efficacy of SO-C101 and anti-PD-1 treatment was not dependent on CD4+ T cells, but mainly on NK and CD8+ T cells. Interestingly, SO-C101 and anti-PD-1 treatment in double NK/CD8+ T cell-depleted mice decreased tumor growth which suggests an involvement of other immune cell populations in the anti-tumor efficacy. SO-C101 stimulated the proliferation and the cytotoxic activity of NK cells and memory CD8+ T cells without significant expansion of regulatory T cells. These data show the importance of various immune cell populations during SO-C101 monotherapy and the treatment in combination with anti-PD-1 antibodies, and set a base for further complex analysis of SO-C101 behavior. The therapeutic potential of SO-C101 is currently being tested in an ongoing Phase I clinical study in cancer patients. Citation Format: Irena Adkins, Romana Mikyskova, Nada Hradilova, Guy de Martynoff, David Bechard, Ulrich Moebius, Milan Reinis, Radek Spisek. SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner [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 6686.

Abstract PR18: A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer

Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of immune evasion in cancer and explore novel therapeutic modalities. Recently, glyco-immune checkpoints have been identified as an important modulator in cancer immune escape. Upon ligation of cancer-specific sialylated glycans, glyco-immune checkpoints suppress multiple facets of cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation anticancer T-cell immunity. However, due to the complexity and heterogeneity of cancer sialylated glycans and the profound species differences of the glyco-immune checkpoints between human and mouse, this immunomodulatory pathway remains unexplored for therapeutic interventions. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), to target this glyco-immune checkpoint axis. EAGLE overcomes the complexity of cancer glycans and specifically cleaves the immunosuppression-mediating key sugar moieties, the terminal sialic acids of various sialylated glycans on tumor cells. We hypothesized that EAGLE could block the immunosuppressive glyco-immune checkpoints in the tumor microenvironment and potentiate innate and adaptive immune responses to treat cancer. Indeed, systematic delivery of an EAGLE molecule decreased the levels of immunosuppressive sialic acids on tumor cells, and increased T-cell infiltration and activation in syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy, and 100% cures in combination with anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced antitumor immunologic memory. In summary, we demonstrated that this novel therapeutic modality, EAGLE, can release glyco-immune checkpoint-mediated immunosuppression in the tumor microenvironment and potentiate innate and adaptive antitumor immunity, which offers a novel and promising immunomodulatory strategy to treat cancer. This abstract is also being presented as Poster A91. Citation Format: Li Peng, Adam Petrone, Lizhi Cao, James Broderick. A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR18.

Combination of NKTR-255, a Polymer Conjugated Human IL-15, with CD19 CAR T Cell Immunotherapy in a Preclinical Lymphoma Model

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member.

Self-Assembled Nanofibers Elicit Potent HPV16 E7-Specific Cellular Immunity And Abolish Established TC-1 Graft Tumor.

BackgroundVaccines are one of the most promising strategies for immunotherapy of HPV associated tumors; however, they generally lack significant clinical efficacy at present. This inefficacy might be due to inefficient generation of anti-tumor cellular immune responses.PurposeThis study aimed to assess the potential of using self-assembled nanofibers as a new vaccine platform to elicit potent HPV antigen - specific anti-tumor immunity.MethodsA HPV16 E744-62 peptide was chemically appended to the N terminus of self-assembling peptide Q11. The nanofibers were prepared and used to immunize mice through a preventive or therapeutic strategy in a TC-1 graft tumor model.ResultsPreventive immunization with nanofibers almost completely suppressed the growth of primarily grafted TC-1 tumors and even a re-challenge of tumor cells after a six-week rest. Therapeutic immunization significantly increased the levels of effector Th1 cells, CTLs and the cytokines IFN-γ and TNF-α in E7 peptide-stimulated splenocytes, and the immunization reduced Th2, MDSC and IL-4 contents compared to the controls. The nanofiber immunization significantly suppressed the growth of established tumors and achieved 66.7% and 50% tumor-free in mice carrying 2–3 mm tumors and even larger tumors with a diameter of 5–6 mm respectively. In addition, the nanofibers were more efficient than the corresponding unassembled peptides for the treatment of established larger size tumors.ConclusionThe results indicated that self-assembling nanofibers could elicit robust HPV antigen -specific anti-tumor cellular immunity and are a potent antigen delivery system for HPV related tumor vaccines.

Abstract 1552: A novel PD1-IL2v immunocytokine for preferential cis-activation of IL-2R signaling on PD-1 expressing T cell subsets strongly potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism

Abstract High-dose IL-2 is approved for patients with metastatic melanoma and renal cell cancer, but is associated with significant toxicity. We have described tumor-targeted CEA-IL2v and FAP-IL2v immunocytokines that are based on an engineered IL2v moiety with abolished binding to IL-2Ra (CD25) to avoid undesired CD25-mediated toxicities and Treg expansion. Their antibodies bind with high affinity to either CEA or FAP, which are broadly expressed in various tumors, and mediate retention/accumulation in malignant lesions. In an effort to further maximize the potency of IL-2R activation of T cells (aka signal 3) without the side effects of wildtype IL-2, we have generated PD1-IL2v that binds to the checkpoint inhibitor PD-1 and delivers IL2v preferentially in cis to PD-1+ T cells substituting binding to CD25. This enables high affinity IL2R signaling selectively in recently primed antigen specific TILs and stem-cell like subsets. Binding/competition experiments demonstrated that PD1-IL2v is 50-fold more potent than FAP-IL2v in inducing p-STAT5 in PD-1 positive T cells, whereas it has equivalent potency on PD-1 negative T cells. Notably, although Tregs can express low constitutive levels of PD-1, PD1-IL2v shows preferential binding to Teff vs. Tregs. Furthermore, PD1-IL2v can overcome Treg suppression of Tconv effector cells to a greater extent than PD-1, FAP-IL2v or their combination. For use in syngeneic mouse models muPD1-IL2v was generated. In an orthotopic pancreatic Panc02-Luciferase model administration of muPD1-IL2v (1 mg/kg IV, qw) resulted in rapid and complete elimination of tumor cells (loss of luciferase signal) and in long term survival (> 140 days) in 7/7 treated animals with protection from tumor cell re-challenge, whereas only 1/7 animals in the muPD-1 + muFAP-IL2v combination group (10 mg/kg + 2.5 mg/kg IV, qw) and none of the animals treated with the respective monotherapies showed long term survival. IHC showed that the improved outcome with muPD1-IL2v was related to a strong intra-tumoral increase of CD3+ CD8+ T cells expressing PD-1 and GrzB. In an immuno-PD study with s.c. Panc02 tumors muPD1-IL2v strongly increased the number of IFNg+ TNFa+ multifunctional and cytotoxic GrzB+ PD-1+ T cells in the tumor accompanied with a 20-fold increase in CD8/CD4 ratio and a strong increase in the CD8+ TEM population resulting in subsequent tumor control or remission in 50% of the animals, respectively. In summary, our data demonstrate that preferential cis-targeting of IL2v to PD-1+ antigen specific T cells with PD1-IL2v results in a strong potentiation of T cell response and anti-tumor efficacy as compared to the combination of PD-1 checkpoint inhibition with FAP-IL2v. These preclinical data establish PD1-IL2v as a promising next generation IL-2 for cancer immunotherapy. Citation Format: Christian Klein, Laura Codarri-Deak, Valeria Nicolini, Stefan Seeber, Laura Lauener, Marine Richard, Esther Bommer, Maria Karagianni, Johannes Sam, Ramona Schlenker, Marisa Mariani, Petra Petra Schwalie, Sylvia Herter, Marina Bacac, Inja Waldhauer, Anne Freimoser-Grundschober, Volker Teichgraeber, Pablo Umana. A novel PD1-IL2v immunocytokine for preferential cis-activation of IL-2R signaling on PD-1 expressing T cell subsets strongly potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism [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 1552.

Abstract 4152: Irreversible electroporation induces immunogenic cell death and mediates durable response in orthotopic PDAC model in combination with anti-PD-1

Abstract Immunotherapy has only limited efficacy against pancreatic ductal adenocarcinoma (PDAC) because of the presence of an immunosuppressive tumor-associated stroma. Here, we showed that combined IRE and anti-PD-1 immune checkpoint blockade significantly suppressed tumor growth and prolonged the lives of immunocompetent mice bearing well-established orthotopic PDAC. Remarkably, more than 35% of mice had a durable response, and were found to be free of residual tumor upon necropsy. All mice that survived for 60 days after IRE and anti-PD1 treatments rejected tumor cell re-challenge. Analyses of splenocytes confirmed that these long-term survivors developed an anti-tumor memory T cell response. Further mechanistic studies unveiled that the efficacy of IRE + anti-PD-1 could be attributed to multiple factors, including rapid release of danger associated molecular patterns, activation of DCs, and alleviation of immunosuppressive tumor microenvironment. Neutralization of CD8+ T cells by anti-CD8 antibody nullified the antitumor effect of combined IRE and anti-PD-1, suggesting that tumor-infiltrating CD8+ T cells played a key role in mediating the anti-tumor efficacy of IRE + anti-PD-1.Given that both IRE and anti-PD-1 antibodies are already used in the clinic, our results support the translation of this combination as a promising approach for treating patients with PDAC. Citation Format: Jun Zhao, Xiaofei Wen, Tingting Li, Marites Melancon, Sanjay Gupta, Weiyi Peng, Chun Li. Irreversible electroporation induces immunogenic cell death and mediates durable response in orthotopic PDAC model in combination with anti-PD-1 [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 4152.

High-Density Lipoprotein-Mimicking Nanodiscs for Chemo-immunotherapy against Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, for which there is no cure. Treatment effectiveness for GBM has been limited due to tumor heterogeneity, an immunosuppressive tumor microenvironment (TME), and the presence of the blood-brain barrier, which hampers the transport of chemotherapeutic compounds to the central nervous system (CNS). High-density lipoprotein (HDL)-mimicking nanodiscs hold considerable promise to achieve delivery of bioactive compounds into tumors. Herein, we tested the ability of synthetic HDL nanodiscs to deliver chemotherapeutic agents to the GBM microenvironment and elicit tumor regression. To this end, we developed chemo-immunotherapy delivery vehicles based on sHDL nanodiscs loaded with CpG, a Toll-like receptor 9 (TLR9) agonist, together with docetaxel (DTX), a chemotherapeutic agent, for targeting GBM. Our data show that delivery of DTX-sHDL-CpG nanodiscs into the tumor mass elicited tumor regression and antitumor CD8+ T cell responses in the brain TME. We did not observe any overt off-target side effects. Furthermore, the combination of DTX-sHDL-CpG treatment with radiation (IR), which is the standard of care for GBM, resulted in tumor regression and long-term survival in 80% of GBM-bearing animals. Mice remained tumor-free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of anti-GBM immunological memory. Collectively, these data indicate that sHDL nanodiscs constitute an effective drug delivery platform for the treatment of GBM, resulting in tumor regression, long-term survival, and immunological memory when used in combination with IR. The proposed delivery platform has significant potential for clinical translation.

Abstract B034: A novel immunomodulatory strategy of targeting glyco-immune checkpoints with EAGLE technology

Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of cancer immune evasion and explore novel therapeutic modalities. Glyco-immune checkpoint axis plays a critical role in modulating innate and adaptive immune responses against cancer. However, this pathway is underexplored for therapeutic interventions of cancer, because the complexity and heterogeneity of glycan-ligands on tumor cells pose grand challenges for conventional therapeutic modalities. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which can overcome the heterogeneity and complexity problems and specifically edit tumor-specific glycans. We evaluated the efficacy of an EAGLE molecule and studied its mechanism of actions in a breast cancer EMT6 syngeneic tumor model. Systematic delivery of the EAGLE molecule decreased the expression levels of immunosuppressive glycan-ligands on tumor cell surfaces, and increased T-cell infiltration and activation in the syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy and 100% cures in combination with an anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced anti-tumor immunologic memory. In summary, the novel therapeutic modality, EAGLE, blocked the glyco-immune checkpoint pathway in the tumor microenvironment and potentiated innate and adaptive antitumor immunity, which offers a novel immunomodulatory strategy to treat cancer. Citation Format: Li Peng. A novel immunomodulatory strategy of targeting glyco-immune checkpoints with EAGLE technology [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 B034.