Potent Antitumor Responses Research Articles

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Simple SummaryDeveloping new therapeutic cancer vaccines is of paramount importance to counteract tumor escape observed after conventional therapies in certain types of cancer. We have previously shown that the combination of two different vaccine platforms, targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. Here, we show that the heterologous prime-boost combination with a protein vaccine and a viral vector vesicular stomatitis virus immunologically reshapes the immune-excluded TC-1 tumor model as well as the inflamed MC-38 tumor model, leading to beneficial therapeutic efficacy. Furthermore, the treatment with a multi-epitope vaccine allowed us to appreciate the various repartition among three antigen-specific cytotoxic T-cell responses combined with the viral boost. The combination leads to improved efficacy in all animals and highlights the importance of combining tumor epitopes. Our vaccine strategy could be further extended to prophylactic cancer vaccines and beyond, for infectious diseases.Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Characterization of Cytotoxic Macrophages in a Pre-Clinical Model of Epstein-Barr Virus (EBV)-Driven Lymphoproliferative Disease

Epstein-Barr Virus (EBV) is an oncogenic herpes virus associated with the development of malignancies associated with poor outcomes. Current treatments lead to further immune suppression, increasing the risk of EBV reactivation and other opportunistic infections. Strategies aimed at reversing the immune-suppressive microenvironment are likely to result in more durable responses while avoiding deleterious effects of chemotherapy. It is well-established that macrophages play an important role in inhibiting the anti-tumor response as evidenced by poor prognosis associated with increasing tumor-associated macrophage (TAM) density or lower ratio of cytotoxic T lymphocytes. Here we describe a pre-clinical model showing the expansion of cytotoxic TAMs and potential mechanisms to prevent this immunosuppressive environment.To assess soluble factors produced by the interaction of tumor on immune effector cells, we used a co-culture (CoCx) system of EBV-transformed human B cell lymphoblastoid cell lines (LCL) cultured with autologous peripheral blood mononuclear cells (PBMC). Serum-free medium collected after 48 hrs showed increased myeloid-associated factors which were absent from either the LCL or PBMC cultures, suggesting the interaction of tumor cells with immune effectors was leading to the production of chemokines known to attract myeloid cells. When purified autologous CD14+ monocytes were incubated in the presence of CoCx conditioned medium (CM) they generated proliferative foci that were absent in the LCL or PBMC CM alone. To better understand which cellular subsets were necessary for the release of these soluble factors, immune subsets were removed from PBMC prior to incubation with LCL or purified and incubated with LCL. We found that T cells, (CD8+ and CD4+), when incubated in the presence of LCL were necessary and sufficient to stimulate the proliferation of myeloid cells. CD56+, CD14+, or CD19+ subsets within the CM did not appear to contribute to the proliferation of purified myeloid cells.We next used flow cytometry to characterize the monocyte-derived population that expanded when introduced to CoCx CM. We again isolated autologous CD14+ monocytes from PMBC and incubated them for 6 days with CoCx CM followed by immunophenotyping. The monocyte-derived population retained CD14 and CD11b in addition to showing pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR, and CD163 respectively), and PD-L1, indicating activation consistent with a TAM phenotype. To characterize the functional consequence of this cell population, CoCx CM-derived macrophages were incubated with autologous T cells that were either activated non-specifically with anti-CD3/C28 or left inactivated. Surprisingly, we noted a marked decrease in viable activated T cells (both CD4+ and CD8+) when exposed to conditioned macrophages. However, the inactivated T cell populations were only mildly effected. Together, with previously described experiments, this suggests a mechanism by which exposure of T cells to EBV+ lymphoblastic tumor lines results in cytokine release, polarizing monocytes to TAMs that, in turn, exhibit cytotoxic activity to tumor-specific T cells. The precise mechanism of cytotoxicity is under investigation.We previously reported the eIF4A translational inhibitor silvestrol potentiated a potent immune-mediated anti-tumor response against EBV-driven lymphoma mainly by promoting expansion and activity of EBV-specific cytotoxic T cells. We next questioned if macrophages were important to this mechanism. We found that with silvestrol, when added to CoCx, the resultant CM did not drive the proliferation of TAMs, and Immunophenotype of CoCx showed silvestrol treatment to result in the ablation of CD14+ cells from culture. Interrogation by transcriptome analysis indicated a potential role of aryl hydrocarbon receptor (AhR). Protein analysis by western blotting indicated an increase of AhR in isolated monocytes when exposed to CoCx CM, which was abrogated by addition of silvestrol. Together these results suggest AhR may play a role in the polarization of macrophages within the tumor microenvironment leading to elimination of tumor-specific T cells. While full mechanistic characterization is ongoing, these preliminary results provide a potentially novel approach to modulate tumor specific immunity in this challenging group of EBV-related malignancies. DisclosuresBaiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Ultrasound-triggered release reveals optimal timing of CpG-ODN delivery from a cryogel cancer vaccine

Recently, several injectable scaffold-based cancer vaccines have been developed that can recruit and activate host dendritic cells (DCs) and generate potent antitumor responses. However, the optimal timing of adjuvant delivery, particularly of the commonly used cytosine-phosphodiester-guanine-oligonucleotide (CpG-ODN), for scaffold-based cancer vaccines remains unknown. We hypothesized that optimally timed CpG-ODN delivery will lead to enhanced immune responses, and designed a cryogel vaccine system where CpG-ODN release can be triggered on-demand by ultrasound. CpG-ODN was first condensed with polyethylenimine and then adsorbed to cryogels. Little adsorbed CpG-ODN was released in vitro. Ultrasound stimulation triggered continuous CpG-ODN release, at an enhanced rate even after ultrasound was turned off, with minimal burst release. In vivo, ultrasound stimulation four days post-vaccination induced a significantly higher antigen-specific cytotoxic T-lymphocyte (CTL) response compared to control mice. Furthermore, ultrasound stimulation at this time point generated a significantly higher IgG2a/c antibody titer than all the groups except ultrasound stimulation eight days post-vaccination. This optimal timing of ultrasound-triggered release coincided with peak DC accumulation in the cryogels. By enabling temporal control of vaccine components through release on-demand, this system is a promising platform to study the optimal timing of delivery of immunomodulatory agents for cancer vaccination.

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.

1032TiP A phase I, first-in-human, study of TILT-123, a tumor-selective oncolytic adenovirus encoding TNFa and IL-2, in participants with advanced melanoma receiving adoptive T-cell therapy with tumor-infiltrating lymphocytes

Adoptive T-cell therapy using tumor-infiltrating lymphocytes (TIL) has demonstrated that potent and long-lasting antitumor responses can be achieved with cellular cancer immunotherapy in solid tumor patients. Yet, the therapy yields various toxicities, due to toxic lymphodepleting preconditioning and IL-2 postconditioning. With the intent of decreasing conditioning-related toxicities, while retaining efficacy, we developed TILT-123 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2), a novel oncolytic adenovirus designed to reinvigorate antitumor T-cells.

P53 Activation Effect in the Balance of T Regulatory and Effector Cell Subsets in Patients With Thyroid Cancer and Autoimmunity.

Carcinomas evade the host immune system by negatively modulating CD4+ and CD8+ T effector lymphocytes through forkhead box protein 3 (FOXP3) positive T regulatory cells’ increased activity. Furthermore, interaction of the programmed cell death 1 (PD1) molecule and its ligand programmed cell death ligand 1 (PDL1) inhibits the antitumor activity of PD1+ T lymphocytes. Immunotherapy has become a powerful strategy for tailored cancer patients’ treatment both in adult and pediatric patients aiming to generate potent antitumor responses. Nevertheless, immunotherapies can generate autoimmune responses. This study aimed to investigate the potential effect of the transformation-related protein 53 (p53) reactivation by a peptide-based inhibitor of the MDM2/MDM4 heterodimer (Pep3) on the immune response in a solid cancer, i.e., thyroid carcinoma frequently presenting with thyroid autoimmunity. In peripheral blood mononuclear cell of thyroid cancer patients, Pep3 treatment alters percentages of CD8+ and CD4+ T regulatory and CD8+ and CD4+ T effector cells and favors an anticancer immune response. Of note that reduced frequencies of activated CD8+ and CD4+ T effector cells do not support autoimmunity progression. In evaluating PD1 expression under p53 activation, a significant decrease of activated CD4+PD1+ cells was detected in thyroid cancer patients, suggesting a defective regulation in the initial activation stage, therefore generating a protective condition toward autoimmune progression.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

Ethanol ablation reliably achieves an anti-metastatic response after modulating tumor acidity and regulatory T cells

<h3>Objectives:</h3> Ethylcellulose (ECE) injections are a low-cost, accessible ablation method for patients with localized tumors. In order to increase the immunogenicity of the tumor microenvironment in triple-negative breast cancers, cyclophosphamide (cyclo) and sodium bicarbonate (bicarb) were used to prime the immune system prior to ECE injections in pre-clinical models of cancer. Cyclo specifically depletes pro-tumor regulatory T cells when once at a low-dose of 200 mg/kg (human equivalent dose of 16 mg/kg). Daily oral bicarb has been shown to reduce tumor pH and enhance infiltration of cytotoxic T cells into the tumor. <h3>Methods:</h3> Immunocompetent BALB/c mice were injected with orthotopic triple-negative (P53-null) breast cancer cell lines (4T1, 67NR). Tumor-bearing mice were treated with combinations of ECE, cyclo, and bicarb. On day 7 after tumor inoculation bicarb at 200 mM was added to the drinking water, on day 9 cyclo at 200 mg/kg IP was given, and on day 10 ECE ablation was performed with an intratumoral injection of 150 µL. Tumor growth rates, overall survival, and metastatic burden after treatment were quantified (n=5-7). To quantify metastatic disease, lungs were harvested at day 60 or earlier if the humane tumor burden limit of 2 cm³ was exceeded. H&E was used to quantify the metastatic burden in whole lung sections. <h3>Results:</h3> In both 4T1 and 67NR pre-clinical models, the combination of ECE+cyclo+bicarb significantly improved survival (P=0.005, P=0.01) and reduced metastases (P=0.02, P=0.003). In the 4T1 tumor model (Fig. 1A), tumors responded following the initial inflammatory response from ablation when primed with cyclo+bicarb resulting in increased overall survival (Fig. 1B). More mice were without evidence of disease at day 60 after receiving ECE+cyclo+bicarb (5/7) than sham+cyclo+bicarb (0/6), ECE alone (0/5), and sham alone (0/6). 4T1 is highly metastatic, shown in representative whole lung H&E (Fig. 1C); however, the addition of ECE to cyclo+bicarb significantly decreased the metastatic burden at death (Fig. 1C, P<0.05). The combination of ECE+cyclo+bicarb resulted in significantly increased survival compared to all other combinations in both 67NR and 4T1 tumor models (P<0.05). <h3>Conclusions:</h3> The addition of cyclo+bicarb to ECE produced a potent systemic anti-tumor response reducing the size of the primary tumors and metastases <i>in vivo.</i>

CAR-NK Cells Effectively Target SARS-CoV-2-Spike-Expressing Cell Lines In Vitro.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating ‘off-the-shelf’ S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Abstract 268: DNA-launched HPV E7 nanoparticle vaccine induces potent anti-tumor cytolytic T-cell responses

Abstract Nanotechnology-based approaches have provided promising tools for development of novel cancer immunotherapeutic immunogens through the decoration of multiple antigens on nanoparticle scaffolds. However, challenges with inducing robust cytolytic T cell responses critical to anti-tumor immunity, as well as purification and scalability, have hampered their clinical translation. To address these challenges, here we present the design and characterization of a DNA-launched nanoparticle vaccine displaying the HPV E7 epitope for inducing anti-tumor immune responses. We designed a nanovaccine construct by scaffolding the HPV E7 epitope with bacterial enzyme lumazine synthase. We demonstrate that through electroporation-facilitated DNA vaccination encoding the antigen-scaffold construct, this nanovaccine self-assembles in vivo into nanoparticles displaying 60 copies of the antigen on its surface. Compared to vaccination with CpG-adjuvanted HPV E7 peptide, the DNA-launched nanovaccine induced significantly greater antigen-specific CD8+ T cell responses upon stimulation with the HPV E7 peptide as well as with the epitope natively expressed on the target tumor cells. In a flank model of TC-1 lung cancer, the DNA-launched nanovaccine achieved complete tumor protection in 70% of mice when administered prophylactically. With therapeutic treatment, the nanovaccine achieved complete tumor regression in 60% of mice. In addition, anti-tumor immunity was retained upon rechallenge over 100 days after initial tumor challenge, supporting the induction of immune memory. These promising results, along with its facile characterization and scalability, support that DNA-launched nanovaccines that self-assemble in vivo can induce significant anti-tumor cellular immunity and are ideally positioned for study in clinical translation. Citation Format: Kevin Liaw, Ziyang Xu, Nicholas Tursi, Neethu Chokkalingam, Daniel Kulp, David Weiner. DNA-launched HPV E7 nanoparticle vaccine induces potent anti-tumor cytolytic T-cell responses [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 268.

Abstract 1513: A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy

Abstract Immunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28-55%. Although lasting and complete responses have been achieved, there is substantial opportunity for improvement. Investigation into the role of CD4+ TIL in this setting remains critically underexplored. CD4+ T cells recognize tumor antigen presented on MHC Class II either directly on tumor cells or indirectly through antigen presenting cells (APCs) and are able to elicit potent anti-tumor responses under the appropriate conditions. Here, we present a case study of a metastatic melanoma patient who received adoptive transfer of a predominantly (88%) CD4+ TIL product. This patient demonstrated a complete response (CR) to therapy despite a lack of detection of IFNg in the infusion product in vitro when these TIL were co-cultured with autologous tumor prior to ACT. Tumor recognition was also absent when CD8+ TIL were isolated and stimulated directly with HLA-matched tumor lines, indicating a lack of recognition of shared melanoma antigens presented on MHC Class I. Longitudinal analysis of the peripheral blood of this patient confirmed that the infused CD4+ TIL persisted after therapy for at least six weeks. Whole exome sequencing (WES) performed on the TIL surgical specimen discovered 88 non-synonymous single nucleotide variants (SNVs) as candidate neoantigens. Predicted binding of the resulting mutant peptides to autologous HLA molecules generated a predominantly MHC Class II restricted profile, with 81.8% of variants capable of MHC Class II presentation and greater than half exclusive to MHC Class II only. CD4+ TIL were screened for tumor antigen recognition by upregulation of OX40 and 41BB after stimulation with autologous APCs loaded with mutant peptides. Nearly half (49.2%) of CD4+ TIL responded to tumor-derived peptides. These CD4+ TIL were then sorted into tumor-reactive and non-reactive subsets for further clonal analysis of phenotype and transcriptional profile (scRNASeq) of these T cells in order to characterize the nature of the CD4+ TIL response to tumor antigen. Overall, thorough interrogation of this patient's case study demonstrated evidence of CD4+ TIL involvement in a complete clinical response after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy. Citation Format: MacLean Hall, Pat Innamarato, Amy Mackay, Holly Branthoover, Alex Alfaro, Carolyn Rich, Allison Richards, Jonathan Hensel, Matthew Beatty, John Mullinax, Amod A. Sarnaik, Shari Pilon-Thomas. A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy [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 1513.

Abstract LB172: APVO442: A bispecific T cell-engaging candidate utilizing the ADAPTIR-FLEXTMplatform technology with unique properties designed for optimal tumor distribution and cytotoxic response against PSMA-expressing solid tumors

Abstract Prostate-Specific Membrane Antigen (PSMA), is a tumor-associated antigen (TAA) that is expressed on prostate cancers, including metastatic castration-resistant prostate cancer (mCRPC). Current chemotherapeutic approaches for mCRPC are challenged by development of resistance resulting in limited clinical benefit. Immuno-oncology therapeutic candidates such as bispecifics re-directing T-cell responses to eliminate tumors, are a promising strategy to overcome the limitations of current approaches and provide benefit to patients with aggressive cancers. Here we present preclinical data demonstrating a potential new approach using low affinity targeting of CD3 and high affinity targeting of PSMA for treatment of a solid tumor cancer.APVO442 is Aptevo's bispecific candidate targeting PSMA and CD3. This candidate was designed to have unique pharmacokinetic and safety properties to potentially maximize potent anti-tumor responses against mCRPC. The APVO442 bispecific T-cell engager uses Aptevo's ADAPTIR-FLEX technology to generate a molecule with low-affinity monovalent CD3 engagement, paired with high-affinity bivalent PSMA binding designed to deliver a highly selective T-cell response at the tumor. The unique engineering of APVO442 reduces the potential of binding to CD3 expressed on peripheral T cells, thus minimizing the potential for on-target toxicity, such as cytokine release, and increasing the potential to deliver the effective concentration of the molecule localized to solid tumors.Preclinical testing demonstrated that APVO442 exhibits optimal manufacturability and functional characteristics for lead candidate selection. Anti-PSMA x anti-CD3 constructs with varying binding strengths to CD3 were evaluated for specificity of CD3 binding, ability to enhance T-cell activation, and ability to elicit T-cell-mediated cytotoxicity against PSMA-expressing tumor targets with varying levels of PSMA expression. APVO442 demonstrated 10-fold reduced binding to CD3 and EC50 compared to the highest affinity constructs tested while retaining equivalent binding to tumor cells expressing various levels of PSMA. The differences in CD3 affinity were associated in a slightly lower EC50 of potency however, the maximal responses for in vitro activation of CD4+ and CD8+ T cells including upregulation of CD25/CD69 expression, proliferation and in vitro tumor lysis were comparable between low and high affinity CD3 constructs. Finally, APVO442 induced reduced levels of multiple cytokines in vitro when compared to high affinity competitor molecules.In vivo, APVO442 elicited robust anti-tumor responses of human PSMA-expressing tumors in a murine xenograft tumor model. The in vivo activity of APVO442 was comparable to high affinity CD3 engaging comparators with similarly measured PK profiles. Additional in vivo characterization of APVO442 is ongoing and continued pre-clinical studies are planned for 2021. Citation Format: Rebecca Gottschalk, Robert E. Miller, Lynda Misher, Michelle H. Nelson, Allison Chunyk, Brian Woodruff, Elizabeth Haglin, Gabriela Hernandez-Hoyos, Peter Pavlik, Catherine McMahan, Hilario J. Ramos, David Bienvenue. APVO442: A bispecific T cell-engaging candidate utilizing the ADAPTIR-FLEXTMplatform technology with unique properties designed for optimal tumor distribution and cytotoxic response against PSMA-expressing solid tumors [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 LB172.

Abstract 68: Activating mucosal-associated invariant T cells (MAITs) induces a broad anti-tumor response and offers a novel target for cancer immunotherapy

Abstract Introduction Mucosal-associated invariant T (MAIT) cells are MR1-resricted innate-like T cells that recognize non-peptide antigens including riboflavin derivates. They can be detected in both primary tumors and metastasis, yet their role in cancer is still unclear. In vitro activated MAIT cells have demonstrated anti-tumor activity suggesting their potential for cancer immunotherapy, but in vivo data is lacking. Experimental procedures We systemically administered a combination of the VitaminB2 synthesis pathway-derived antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) with Toll-like receptor 9 agonist CpG for in vivo activation and expansion of MAIT cells. We used three different murine models of liver tumors, a lung metastases model and subcutaneous tumors across two different mouse strains, to assess anti-tumor activity of 5-OP-RU + CpG. Tumor growth over time was measured by in vivo bioluminescence imaging. Phenotypic changes of hepatic, pulmonary and tumor-infiltrating MAIT cells were detected by FLOW cytometry using MR1-tetramers. Immune cell monitoring was performed to identify alterations in the tumor microenvironment. A series of pharmacological depletion experiments was conducted to identify additional effector cells. Results Here, to the best of our knowledge, we report first evidence of MAIT cell-mediated anti-tumor function in vivo when activated by a combination of 5-OP-RU and TLR 9 agonist CpG. Co-administration of 5-OP-RU and CpG induced a strong systemic in vivo expansion and activation of MAIT cells with high expression of activation makers like CD69, pronounced effector memory phenotype and upregulated effector molecules including interferon (IFN)-γ, granzyme B and perforin. MAIT-directed 5-OP-RU + CpG showed pronounced and consistent anti-tumor activity in all tumor models and prolonged mouse survival. Importantly, such tumor inhibition was absent in MAIT-deficient MR1-/- mice. Additional depletion studies showed that NK cells partially mediate the MAIT-induced tumor suppression. Conclusion Increasing evidence suggests that MAIT cells are important players in cancer immunology. MAITs undergo a phenotypic switch and massively expand in vivo upon 5-OP-RU + CpG treatment. These licensed and educated innate-like T cells can then mediate potent anti-tumor responses in murine models and represent an attractive novel target for cancer immunotherapy. We provide a framework for how TCR-dependent pathogenic role of MAITs in malignancies can be overcome using stimulatory agents. Citation Format: Benjamin Ruf, Simon Wabitsch, Chi Ma, Laurence P. Diggs, Bernd Heinrich, Vanessa V. Catania, Varun Subramanyam, Linda Cui, Shunsuke Sakai, Sangmi Oh, Clifton E. Barry, Daniel L. Barber, Tim F. Greten. Activating mucosal-associated invariant T cells (MAITs) induces a broad anti-tumor response and offers a novel target for cancer immunotherapy [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 68.

Rigosertib elicits potent anti-tumor responses in colorectal cancer by inhibiting Ras signaling pathway

BackgroundThe therapeutic potency of Rigosertib (RGS) in the treatment of the myelodysplastic syndrome has been investigated previously, but little is known about its mechanisms of action. MethodsThe present study integrates systems and molecular biology approaches to investigate the mechanisms of the anti-tumor effects of RGS, either alone or in combination with 5-FU in cellular and animal models of colorectal cancer (CRC). ResultsThe effects of RGS were more pronounced in dedifferentiated CRC cell types, compared to cell types that were epithelial-like. RGS inhibited cell proliferation and cell cycle progression in a cell-type specific manner, and that was dependent on the presence of mutations in KRAS, or its down-stream effectors. RGS increased both early and late apoptosis, by regulating the expression of p53, BAX and MDM2 in tumor model. We also found that RGS induced cell senescence in tumor tissues by increasing ROS generation, and impairing oxidant/anti-oxidant balance. RGS also inhibited angiogenesis and metastatic behavior of CRC cells, by regulating the expression of CD31, E-cadherin, and matrix metalloproteinases-2 and 9. ConclusionOur findings support the therapeutic potential of this potent RAS signaling inhibitor either alone or in combination with standard regimens for the management of patients with CRC.

Attenuated Interferon-α Targeted to CD38 Expressing Multiple Myeloma Tumor Cells Induces Robust and Durable Anti-Tumor Responses through Direct Anti-Proliferative Activity in Addition to Indirect Recruitment and Activation of M1 Macrophages

Despite recent advances in treatment, multiple myeloma (MM) remains an incurable disease. We previously reported that targeting an attenuated form of interferon-alpha (IFNα) directly to MM tumor cells via an anti-CD38 antibody induces robust and durable responses in murine xenograft MM models, eliminating large established human tumors in mice. We demonstrated that this attenuated immunocytokine, or Attenukine™, exhibits 10,000-fold increased specificity for CD38-positive cells in vitro compared to native IFNα and, importantly, is less toxic than native IFNα to normal bone marrow cells in vitro. Application of this therapy is predicted to significantly expand the therapeutic index of IFNα therapy beyond that of an immunocytokine, providing a rationale for clinical testing in MM.In this study, we explored potential mechanisms mediating the robust anti-MM tumor activity of the CD38-Attenukine™. In MM xenograft models, immunohistochemistry analysis of ex vivo tumors taken from treated and untreated mice revealed rapid and robust infiltration of M1 (iNOS positive) macrophages in treated MM tumors (shown in figure). Targeted depletion of macrophages in tumor bearing mice prior to treatment demonstrated the importance of macrophages for the robust and durable activity. Follow-on experiments compared the anti-tumor activity of the CD38-Attenukine™ in different mouse strains with varied degrees of immune competence. While treatment of SCID mice bearing NCI-H929 tumors with 5 mg/kg CD38-Attenukine™ eliminated tumors in all mice, tumors in only 2 of 10 mice were eliminated in the NOD-SCID background, and no tumors were eliminated in the NSG mouse strain. The NOD-SCID and NSG strains, which have defective macrophage populations, showed significantly less macrophage infiltration than was observed in the SCID mouse strain. Both the macrophage infiltration and antitumor activity of the CD38-Attenukine™ could be fully restored in NOD-SCID mice when co-treated with an anti-human CD47 antibody. Blocking CD47 signaling is thought to correct the defect in the NOD-SCID macrophage compartment. Interestingly, tumor sections from CD38-Attenukine™ treated mice were caspase-3 negative, suggesting that apoptosis may not be the major mechanism of tumor cell death in these models. In fact, based on the requirement for a glycosylated IgG Fc for robust and durable response, we predict that antibody-dependent cellular phagocytosis (ADCP) is a major mechanism for the observed tumor regression. Additional findings suggest that IFNα-induced chemokine production from tumor cells may signal M1 macrophage infiltration and activation following treatment. In conclusion, the potent anti-tumor response observed in CD38-Attenukine™ treated mice is the result of at least two complementary mechanisms, direct anti-proliferative activity of IFNα on tumor cells and indirect recruitment and activation of tumoricidal macrophages potentially through a chemokine gradient. These findings provide a solid framework supporting clinical evaluation of a CD38-Attenukine™ immunotherapy in MM patients. [Display omitted] DisclosuresPogue:Teva Pharmaceuticals: Employment. Bi:Teva Pharmaceuticals: Employment. Armanini:Teva Pharmaceuticals: Employment. Fatholahi:Teva Pharmaceuticals: Employment. Taura:Teva Pharmaceuticals: Employment. Valencia:Teva Pharmaceuticals: Employment. Yun:Teva Pharmaceuticals: Employment. Sho:Teva Pharmaceuticals: Employment. Jamin:Teva Pharmaceuticals: Employment. Nock:Teva Pharmaceuticals: Employment. Wilson:Teva Pharmaceuticals: Employment.

AGING AND ORAL CANCER DEVELOPMENT

Background Cell aging or cellular senescence is a potent antitumor response characterized by an irreversible cell growth arrest. Paradoxically, aging is considered a risk factor for the development of cancer, with compelling evidence of senescent cells acting as cancer promoters. Senescent cells remain metabolically active and adopt a proinflammatory phenotype known as senescence-associated secretory phenotype (SASP) characterized by the secretion of different factors including IL-6 and IL-8, 2 well-known cancer-associated cytokines. Objective To study the senescence response of normal and dysplastic (OD) oral keratinocytes and to explore pharmacological ways to reduce its potential oncogenic effects. Methods Primary normal and dysplastic keratinocytes were grown until senescence was achieved and changes in gene and protein expression were analyzed. Senescent cells were treated with 2 different drugs (a Rho kinase and cGAS inhibitor) to explore possible ways to modify the SASP. To understand mechanisms regulating senescence, OD cells were genetically modified using the CRISPR/Cas9 system. Results Senescent normal oral keratinocytes and ODs develop a SASP characterized by a significant increase of IL-1α, IL-1β, IL-6, and IL-8, which is accompanied by a decrease in the IL-1 receptor antagonist (IL-1RA). Rock inhibitors are successful in reducing cytokine secretion by senescent cells, whereas cGAS inhibitors can only do it partially. Knock-out of IL-1RA suggests that IL-1RA has important functions in the regulation of senescence. Conclusions Our study shows that senescent oral keratinocytes adopt a proinflammatory state, rich in cytokines known to have oncogenic effects, which can be pharmacologically modified without affecting the senescence response.

Resetting the tumor microenvironment to favor anti-tumor immunity after local ablation.

2561 Background: Percutaneous tumor ablation is a non-surgical method of tumor destruction that leaves necrotic tumor debris in situ. Tumor associated antigens released after ablation have the potential to initiate a systemic anti-tumor immune response, however the hostile tumor microenvironment hinders antigen presentation and T cell activity. We hypothesized that resetting the tumor microenvironment with oral sodium bicarbonate to decrease tumor acidity and low-dose cyclophosphamide to deplete pro-tumor immune cells would improve the ability of ablation to initiate anti-tumor immunity. Methods: Tumor growth, overall survival, and metastatic burden was assessed in orthotopic tumor models of triple-negative breast cancer (67NR, 4T1, and E0771). Tumor ablation was performed on palpable tumors using percutaneous ethanol injection (PEI) with 6% ethylcellulose to improve retention in the tumor. Surgical excision was used as a negative control to test the role of in situ tumor debris. Before ablation mice were placed on 200 mM of sodium bicarbonate (SB) in their drinking water and received a single intraperitoneal injection of 200 mg/kg of cyclophosphamide (CP). Mice surviving to 60 days after tumor implant without a primary tumor or signs of metastases were considered "cured" and re-challenged with 50e5 tumor cells in the contralateral mammary pad. T cell dependance was assessed with in vivo CD8 depletions. Results: The combination of PEI+SB+CP produced a potent anti-tumor response, curing a majority of mice (5/7 of E0771, 8/12 of 67NR, 7/12 of 4T1). No mice were cured using PEI alone, SB alone, CP alone, or any combination of two therapies (0/51 of E0771, 0/73 of 67NR, 0/75 of 4T1,). Re-challenge tumor growth was hindered in mice cured with PEI+SB+CP. Mice receiving PEI+SB+CP had significantly less metastases and lived longer than mice receiving surgical excision alone or surgical excision with SB+CP. Additionally the anti-metastatic response of PEI+SB+CP was undone when CD8+ T cells were depleted. Conclusions: Here the anti-tumor response of local ablation produced by PEI was enhanced by priming the tumor with low-dose CP and oral SB in metastatic breast cancer. These results suggest that tumor ablation with CP and SB can create a T cell dependent, personalized immune response to a tumor using only low-cost, easily accessible supplies, and the host’s own tumor.

Apatinib combined with PD-L1 blockade synergistically enhances antitumor immune responses and promotes HEV formation in gastric cancer.

Apatinib, an antiangiogenic drug, has shown beneficial effects only in a fraction of advanced gastric cancer (GC) patients. Given the recent success of immunotherapies, combination of apatinib with immune checkpoint inhibitor may provide sustained and potent antitumor responses. Immunocompetent mice with subcutaneous MFC tumors grown were given a combination of apatinib and anti-PD-L1 antibody therapy. GC tissues from patients undergoing curative resection in China were collected, and the density of HEVs, MSI status and tumor-infiltrated lymphocytes were analyzed by immunohistochemical staining. Combined apatinib and PD-L1 blockade therapy synergistically delayed tumor growth and increased survival in MFC-bearing immunocompetent mice. The combination therapy promoted antitumor immunity by increasing the ratio of CD8+ cytotoxic T cells to Foxp3+ Treg cells, the accumulation of CD20+ B cells and the Th1/Th2 cytokine ratio (IFN-γ/IL-10). The combination therapy induced the formation of HEVs through activation of LTβR signaling, thus promoting CD8+ cytotoxic T cell and CD20+ B cell infiltration in tumors. In clinical GC samples, the density of HEVs positively correlated with the intratumoral infiltration of CD8+ cytotoxic T cells and CD20+ B cells. MSI-high GC showed a higher density of HEVs, CD8+ cytotoxic T cells and CD20+ B cells than MSS/MSI-low GC. GC patients with high densities of HEVs, CD8+ cytotoxic T cells and CD20+ B cells had an improved prognosis with superior overall survival. Combining apatinib with PD-L1 blockade treatment synergistically enhances antitumor immune responses and promotes HEV formation in GC.

New generation of DNA-based immunotherapy induces a potent immune response and increases the survival in different tumor models

BackgroundStrategies to increase nucleic acid vaccine immunogenicity are needed to move towards clinical applications in oncology. In this study, we designed a new generation of DNA vaccines, encoding an engineered...

19O Preliminary safety, antitumor activity and pharmacodynamics results of HIT-IT MEDI1191 (mRNA IL-12) in patients with advanced solid tumours and superficial lesions

IL-12 is a strong driver of a TH1 immune response, but systemic recombinant IL-12 is poorly tolerated clinically. MEDI1191 (IL-12 mRNA) is a lipid nanoparticle (LNP)-formulated therapy developed for intratumoral (IT) injection, designed to drive local IL-12 production and induce anenestic anti-tumor activity. In preclinical models, IT IL-12 mRNA induces a potent TH1-mediated antitumor response, that is further enhanced by PD-L1 blockade.