Anti-tumor Immunity In Pre-clinical Models Research Articles

LSD1 inhibition improves efficacy of adoptive T cell therapy by enhancing CD8+ T cell responsiveness

The lysine-specific histone demethylase 1 A (LSD1) is involved in antitumor immunity; however, its role in shaping CD8 + T cell (CTL) differentiation and function remains largely unexplored. Here, we show that pharmacological inhibition of LSD1 (LSD1i) in CTL in the context of adoptive T cell therapy (ACT) elicits phenotypic and functional alterations, resulting in a robust antitumor immunity in preclinical models in female mice. In addition, the combination of anti-PDL1 treatment with LSD1i-based ACT eradicates the tumor and leads to long-lasting tumor-free survival in a melanoma model, complementing the limited efficacy of the immune or epigenetic therapy alone. Collectively, these results demonstrate that LSD1 modulation improves antitumoral responses generated by ACT and anti-PDL1 therapy, providing the foundation for their clinical evaluation.

Abstract 2921: Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models

Abstract Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis induced by radiotherapy (RT) and chemotherapy. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved locoregional control at 18 months, 3-year progression-free survival and nearly doubled 5-year overall survival vs. placebo + CRT (53% vs. 28%). Two phase 3 trials, in which patients receive 6 cycles of xevinapant or placebo monotherapy (3 cycles in combination with standard-of-care (C)RT followed by 3 cycles of monotherapy), are ongoing. Based on the roles of IAPs in apoptosis and tumor immunity, continual dosing of xevinapant post RT may deliver additional therapeutic benefit through modulation of multiple tumor microenvironment (TME) compartments. Methods: The impact on antitumor efficacy of xevinapant dosing duration was evaluated in A1419 (head and neck), LLC (lung) and MC38 (colorectal) syngeneic tumor-bearing mice treated with vehicle control, RT alone (3.6Gy, QD, 5 days), or RT + xevinapant (100 mg/kg, QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation in MC38 tumors, RNAseq and FACS-based immune profiling was performed, followed by functional validation with ex vivo ELISpot assays, in vitro immunogenic cell death assays, T cell activation assays, and macrophage polarization and viability assays. To evaluate the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation and CAF-modulated immune phenotype, activation assay and cytokine profiling was performed. Results: In the tumor models tested, RT in combination with extended xevinapant dosing markedly improved efficacy and prolonged survival compared to treatment arms of RT with shorter xevinapant dosing durations, RT alone, and vehicle control. RNAseq and FACS analyses suggested a TME with enhanced antitumor immunity, while ELISpot and in vitro T cell assays confirmed that extended xevinapant dosing promoted antigen-specific T cell response and T cell activation. Furthermore, macrophage polarization and viability assays suggested that xevinapant reduced the viability of M2 macrophages which may have contributed to the increased M1:M2 macrophage ratio and the improved antitumor immune response observed in vivo. In addition, via the suppression of RT-mediated CAF activation, xevinapant may promote M1 polarization of macrophages and recruitment of T cells by upregulating GM-CSF and CXCL10 in CAFs. Conclusions: Extended dosing of xevinapant post concurrent RT improved antitumor efficacy and prolonged survival of tumor-bearing mice. Mechanistic investigation suggested that such benefit may be, in part, modulated by xevinapant enhanced antitumor immunity. Citation Format: Tsz-Lun Yeung, Feng Jiang, Hui Huang, Huakui Yu, Li-Ya Chiu, Mathilde Bonnemaison, Bo Marelli, Roberta Ferretti, Ralph Lindemann, Christina Esdar. Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2921.

Blockade of EP4 by ASP7657 Modulates Myeloid Cell Differentiation In Vivo and Enhances the Antitumor Effect of Radiotherapy.

The tumor microenvironment (TME) is thought to influence the antitumor efficacy of immuno-oncology agents through various products of both tumor and stromal cells. One immune-suppressive factor is prostaglandin E2 (PGE2), a lipid mediator whose biosynthesis is regulated by ubiquitously expressed cyclooxygenase- (COX-) 1 and inducible COX-2. By activating its receptors, PGE2 induces immune suppression to modulate differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) rather than dendritic cells (DCs). Pharmacological blockade of prostaglandin E receptor 4 (EP4) causes a decrease in MDSCs, reprogramming of macrophage polarization, and increase in tumor-infiltrated T cells, leading to enhancement of antitumor immunity in preclinical models. Here, we report the effects of the highly potent EP4 antagonist ASP7657 on the DC population in tumor and antitumor immune activation in an immunocompetent mouse tumor model. Oral administration of ASP7657 inhibited tumor growth, which was accompanied by an increase in intratumor DC and CD8+ T cell populations and a decrease in the M-MDSC population in a CT26 immunocompetent mouse model. The antitumor activity of ASP7657 was dependent on CD8+ T cells and enhanced when combined with an antiprogrammed cell death-1 (PD-1) antibody. Notably, ASP7657 also significantly enhanced the antitumor efficacy of radiotherapy in an anti-PD-1 antibody refractory model. These results indicate that the therapeutic potential of ASP7657 arises via upregulation of DCs and subsequent CD8+ T cell activation in addition to suppression of MDSCs in mouse models and that combining EP4 antagonists with radiotherapy or an anti-PD-1 antibody can improve antitumor efficacy.

Abstract 610: The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma

Abstract Blocking inhibitory immune receptors has shown limited clinical benefit in patients with multiple myeloma (MM), warranting the discovery of alternative immune inhibitory pathways. Blockade of the immune checkpoint TIGIT was shown to enhance anti-tumor immunity in MM pre-clinical models. In addition to TIGIT, the DNAM1 axis includes the novel inhibitory receptor PVRIG. Both TIGIT and PVRIG deliver inhibitory signals to T and NK cells and compete with the co-activating receptor DNAM1 for binding to PVR and PVRL2, respectively. Accordingly, TIGIT and PVRIG co-blockade was shown to synergize in enhancing anti-tumor immunity in preclinical models. PVRL2 and PVR were shown to be expressed on MM plasma cells in the bone marrow (BM). In this study, we evaluated DNAM1 axis receptor expression in the BM of patients with MM. BM mononuclear cells derived from 21 patients with MM were analyzed for the expression of PD1 and DNAM1 axis molecules by flow cytometry. Patients classified with progressed disease (PD), or complete response (CR) were treated with multiple lines of therapies including targeted therapies, chemotherapies, proteasome inhibitors. PVRIG demonstrated the highest expression among all evaluated receptors on NK (88%), NKT (81%) and CD8+ T cells (79%), significantly higher than PD1 (p<0.0001) and TIGIT (P<0.001). TIGIT showed substantially increased expression on NK (64%, P<0.0001) and CD8+ T cells (58%, p<0.05) compared to PD1 (expressed on 12% of NK cells and 42% of CD8+ T cells). Importantly, 50% and 60% of CD8+ and NK cells, respectively, co-expressed TIGIT and PVRIG, and all examined cell populations showed increased levels of DNAM1 (>50%). Additionally, the expression of PVRIG ligand, PVRL2, on BM plasma cells from 6 patients with MM was demonstrated by immuno-histochemistry and flow cytometry. A fraction of CD8+ T cells were DNAM1- and positive for PVRIG/TIGIT/PD1 (22%), suggesting the accumulation of an exhausted CD8+ T cell population in the MM tumor microenvironment. PVRIG showed significantly higher (p<0.0001) expression on DNAM+ CD8+ T cells (81%), compared to TIGIT (43%) and PD1 (34%), supporting the potential of PVRIG blockade to enhance DNAM1 signaling and subsequent CD8+ T cell activation. Finally, patients with CR had a trend towards higher DNAM1 expression on CD8+ T cells (75%) compared to PD patients (54%, p=0.057). To conclude, DNAM1 axis receptors are dominantly expressed on MM BM lymphocytes, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM1 in patients with PD suggests a link between DNAM1 axis and clinical outcome. Our findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT and suggest that combined blockade of these checkpoints may potentially benefit patients with MM, placing the DNAM1 axis as a promising therapeutic pathway in MM therapy. Citation Format: Masha Frenkel, Zoya Alteber, Ning Xu, Mingjie Li, Haiming Chen, Gady Cojocaru, James Berenson, Eran Ophir. The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma [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 610.

SENTI-101, a Preparation of Mesenchymal Stromal Cells Engineered to Express IL12 and IL21, Induces Localized and Durable Antitumor Immunity in Preclinical Models of Peritoneal Solid Tumors

Advanced peritoneal carcinomatosis including high-grade ovarian cancer has poor prognoses and a poor response rate to current checkpoint inhibitor immunotherapies; thus, there is an unmet need for effective therapeutics that would provide benefit to these patients. Here we present the preclinical development of SENTI-101, a cell preparation of bone marrow-derived mesenchymal stromal (also known as stem) cells (MSC), which are engineered to express two potent immune-modulatory cytokines, IL12 and IL21. Intraperitoneal administration of SENTI-101 results in selective tumor-homing and localized and sustained cytokine production in murine models of peritoneal cancer. SENTI-101 has extended half-life, reduced systemic distribution, and improved antitumor activity when compared with recombinant cytokines, suggesting that it is more effective and has lower risk of systemic immunotoxicities. Treatment of tumor-bearing immune-competent mice with a murine surrogate of SENTI-101 (mSENTI-101) results in a potent and localized immune response consistent with increased number and activation of antigen presenting cells, T cells and B cells, which leads to antitumor response and memory-induced long-term immunity. Consistent with this mechanism of action, co-administration of mSENTI-101 with checkpoint inhibitors leads to synergistic improvement in antitumor response. Collectively, these data warrant potential clinical development of SENTI-101 for patients with peritoneal carcinomatosis and high-grade ovarian cancer.Graphical abstract: SENTI-101 schematic and mechanism of actionSENTI-101 is a novel cell-based immunotherapeutic consisting of bone marrow-derived mesenchymal stromal cells (BM-MSC) engineered to express IL12 and IL21 intended for the treatment of peritoneal carcinomatosis including high-grade serous ovarian cancer. Upon intraperitoneal administration, SENTI-101 homes to peritoneal solid tumors and secretes IL12 and IL21 in a localized and sustained fashion. The expression of these two potent cytokines drives tumor infiltration and engagement of multiple components of the immune system: antigen-presenting cells, T cells, and B cells, resulting in durable antitumor immunity in preclinical models of cancer.

Abstract 1021: Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening

Abstract Genomic instability in cancer cells leads to cellular stress through the accumulation of aberrant nucleic acid species in the cytosol. We have shown that PARP7, a monoPARP, is a negative regulator of cytosolic nucleic acid sensing in cancer cells. RBN-2397 is a potent and selective PARP7 inhibitor that induces antitumor immunity in preclinical models and is currently being evaluated in a Phase I clinical trial. In our preclinical investigations, we found that in a subset of cancer cell lines, such as NCI-H1373, inhibition of PARP7 triggers Type I IFN release, STAT1 phosphorylation, and growth arrest. In contrast, other cell lines, for example, HARA, do not mount an IFN-response upon PARP7 inhibition, even though they are responsive to transfection of exogenous nucleic acids and PARP7 is expressed and enzymatically active. To investigate the underlying mechanism of PARP7 inhibition and to determine the drivers of the differential sensitivity across cell lines we performed arrayed CRISPR knockout screens, targeting approximately 240 genes in the nucleic acid sensing and IFN signaling pathways, in the presence and absence of PARP7 inhibition. Our arrayed screens confirmed multiple hits from a previous genome-wide pooled synthetic/lethal CRISPR dropout screen. For example, targeting genes in the cGAS/STING pathway conferred resistance to PARP7 inhibition in the NCI-H1373 responder cells, suggesting a critical dependence on this sensing pathway. In the PARP7 inhibitor-resistant HARA cells, deletion of components of innate immune-signaling (such as AIM2 and ADAR1), the NF-κB pathway, and genes involved in autophagy sensitized the cells to PARP7 inhibition. We further delineated the function of PARP7 by comparing the effects of the CRISPR perturbation across different cellular readouts such as STAT1 phosphorylation, IFN release, and proliferation. With our work, we shed light on the mechanism by which PARP7 acts as a critical suppressor of the innate immune response. Our findings demonstrate both redundancy and crosstalk between different nucleic acid-sensing pathways and may explain why some cell lines are resistant to PARP7 inhibition. Citation Format: Bin Gui, Ryan Abo, Patrick Flynn, Alvin Z. Lu, Jan-Rung Mo, Joseph M. Gozgit, Melissa M. Vasbinder, Zacharenia A. Varsamis, Andrew Santospago, Victoria M. Richon, Kevin W. Kuntz, Heike Keilhack, Timothy J. Mitchison, Mario Niepel. Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening [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 1021.

Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models.

Increasing clinical evidence has demonstrated that the deletion or mutation of tumor suppressor genes such as the gene-encoding phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in cancer cells may correlate with an immunosuppressive tumor microenvironment (TME) and poor response or resistance to immune checkpoint blockade (ICB) therapy. It is largely unknown whether the restoration of functional PTEN may modulate the TME and improve the tumor's sensitivity to ICB therapy. Here, we demonstrate that mRNA delivery by polymeric nanoparticles can effectively induce expression of PTEN in Pten-mutated melanoma cells and Pten-null prostate cancer cells, which in turn induces autophagy and triggers cell death-associated immune activation via release of damage-associated molecular patterns. In vivo results illustrated that PTEN mRNA nanoparticles can reverse the immunosuppressive TME by promoting CD8+ T cell infiltration of the tumor tissue, enhancing the expression of proinflammatory cytokines, such as interleukin-12, tumor necrosis factor-α, and interferon-γ, and reducing regulatory T cells and myeloid-derived suppressor cells. The combination of PTEN mRNA nanoparticles with an immune checkpoint inhibitor, anti-programmed death-1 antibody, results in a highly potent antitumor effect in a subcutaneous model of Pten-mutated melanoma and an orthotopic model of Pten-null prostate cancer. Moreover, the combinatorial treatment elicits immunological memory in the Pten-null prostate cancer model.

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation

BackgroundPreclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation.MethodsWe developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor.ResultsOur data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor.ConclusionsWe have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8+ T Cells

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ Tcells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

ICOS agonism by JTX-2011 (vopratelimab) requires initial T cell priming and Fc cross-linking for optimal T cell activation and anti-tumor immunity in preclinical models.

Immunotherapy checkpoint inhibitors, such as antibodies targeting PD-1 and CTLA-4, have demonstrated the potential of harnessing the immune system to treat cancer. However, despite encouraging results particularly with respect to survival, only a minority of patients benefit from these therapies. In clinical studies aimed at understanding changes in the immune system following immunotherapy treatment, ICOS (Inducible T cell CO-Stimulator) was shown to be significantly up-regulated on CD4+ T cells and this was associated with clinical activity, indicating that ICOS stimulatory activity may be beneficial in the treatment of solid tumors. In this report, we describe the generation of specific, species cross-reactive, agonist antibodies to ICOS, including the humanized clinical candidate, JTX-2011 (vopratelimab). Preclinical studies suggest that the ICOS stimulating antibodies require Fc receptor cross-linking for optimal agonistic activity. Notably, the ICOS antibodies do not exhibit superagonist properties but rather require T cell receptor (TCR)-mediated upregulation of ICOS for agonist activity. Treatment with the ICOS antibodies results in robust anti-tumor benefit and long-term protection in preclinical syngeneic mouse tumor models. Additional benefit is observed when the ICOS antibodies are administered in combination with anti-PD-1 and anti-CTLA-4 therapies. Based on the preclinical data, JTX-2011 is currently being developed in the clinical setting for the treatment of solid tumors.

Abstract 5830: Senolytic drug ABT-263 enhances cisplatin- and TIL-induced cell death in vitro but has limited in vivo activity in preclinical models of head and neck cancer

Abstract Background/Objectives: Cisplatin and other cytotoxic chemotherapy drugs may induce senescence, rather than cell death, at sublethal doses. We hypothesized that the senolytic drug ABT-263 (navitoclax) might enhance cisplatin cytotoxicity and anti-tumor immunity in preclinical models of head and neck squamous cell carcinoma (HNSCC). Methods/Results: When human HNSCC cell lines were treated with sublethal doses of cisplatin for 72 hours, they expressed increased levels of senescence markers p16 and beta-galactosidase. In additional experiments, cells were rendered senescent with cisplatin and then treated for another 72 hours with navitoclax, which enhanced cisplatin-induced cell death. Three different cisplatin-sensitive syngeneic mouse models were then used to investigate whether the addition of navitoclax can enhance cisplatin-induced tumor cell death in vivo. Mice were treated with weekly cycles consisting of cisplatin on day 1 (5-6 mg/kg IP) followed by navitoclax on days 3, 4, and 5 (100 mg/kg, OG). The addition of navitoclax caused an early, very slight improvement in tumor growth delay in the TC-1 model, but not in the MEER or MOC1 models. We next investigated whether navitoclax might enhance the anti-tumor immune effects of cisplatin and/or tumor infiltrating leukocytes (TIL) ex vivo. Naive tumors were harvested and TIL were cultured with IL-2 for 10-14 days, then sorted. Murine tumor cells were then cultured with cisplatin, navitoclax, and/or TIL. In the TC-1 and MOC1 models, but not in the MEER model, navitoclax modestly enhanced tumor-cell killing by TIL. Tumor cell killing was greatest with cisplatin, navitoclax, and TIL in combination. We then investigated whether navitoclax might enhance the anti-tumor activity of cisplatin and anti-PD-1 in vivo. With twice-weekly anti-PD-1 antibody alone or added to the cisplatin and/or navitoclax in the TC-1 model, no additional anti-tumor activity was noted; flow cytometry of naïve TC-1 tumors showed extremely low numbers of CD8+ T cells as a possible explanation. Similar experiments in the MOC1 model, along with mechanistic experiments to investigate resistance to navitoclax in vivo, are in progress. Conclusions: Navitoclax enhances cisplatin-induced death of human HNSCC cells in vitro and enhances the killing of murine tumor cells when cultured with TIL ex vivo. However, in multiple syngeneic mouse models, navitoclax failed to enhance anti-tumor activity of cisplatin or anti-PD-1 checkpoint blockade. Possible mechanisms of navitoclax resistance in vivo may include enhanced trafficking of immunosuppressive cells to the tumor microenvironment following secretion of senescence-associated cytokines by cisplatin-treated tumor cells. Supported by NIDCD intramural project number ZIA-DC000090. Citation Format: Youji Hong, Sreenivasulu Gunti, Yvette Robbins, Nicole C. Schmitt. Senolytic drug ABT-263 enhances cisplatin- and TIL-induced cell death in vitro but has limited in vivo activity in preclinical models of head and neck cancer [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 5830.

Abstract 893: A chimeric PD1-CD28 switch receptor enhances the activity of TRuC-T cells

Abstract Cluster of differentiation 28 (CD28) and programmed death receptor 1 (PD-1) are members of the CD28 superfamily of co-receptors that have critical roles in the regulation of T cell-mediated immunity and inflammation. Ligation of CD28 synergizes with T cell receptor (TCR) signaling to enhance T cell activation through the PI3K-Akt pathway, while PD-1 ligation by its ligands (PD-L1/L2) sequesters critical mediators of signaling from the TCR complex, thereby shunting T cell activation and effector function. Thus, the expression of PD-L1/L2 in solid tumors may pose a significant barrier to anti-tumor immunity and the efficacy of adoptive T cell therapies (ACT). We have recently described a novel class of engineered T cells that integrate a T cell receptor fusion construct (TRuC®) into the natural TCR complex, thereby reprogramming the specificity of the T cell to recognize tumor surface antigen in a human leukocyte antigen (HLA)-independent fashion. TC-210 T cells expressing mesothelin (MSLN) specific TRuCs demonstrate robust anti-tumor immunity in preclinical models of mesothelioma, protecting mice from tumor re-challenge while inducing lower levels of inflammatory cytokine release when compared to a 2nd generation MSLN-targeted CAR T. Here, we show that co-expression of a PD-1:CD28 switch receptor comprising the PD-1 extracellular domain fused to the CD28 intracellular domain, enhances the activity of TC-210 T cells. When compared to TC-210 expressing only the TRuC, co-expression of PD1:CD28 was able to restore PD-L1 mediated inhibition of cytokine production and proliferation in co-culture with tumor cells. In vivo and molecular mechanistic studies are currently underway. Citation Format: Derrick P. McCarthy, Sarah Guyette, Michael Lofgren, Jyothi Sethuraman, Thamara DeSilva, Ahmar Aziz, Troy Patterson, Shruti Datari, Tiffany Chan, Philippe Kieffer-Kwon, Christopher J. Rold, Reshma Singh, Jian Ding, Holly Horton, R. Anthony Barnitz, Andrew Cornforth, Robert Tighe, Robert J. Hofmeister, Dario A. Gutierrez. A chimeric PD1-CD28 switch receptor enhances the activity of TRuC-T cells [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 893.

Immunohistochemistry analyses of LAG-3 expression across different tumor types and co-expression with PD-1.

e15086 Background: Concomitant blockade of LAG-3 and PD-1 has been shown to restore T-cell activation and enhance anti-tumor immunity in preclinical models. Clinical trials evaluating anti-LAG-3/anti-PD-1 mAb combinations or bispecific molecules are ongoing. Establishing the tumor expression profile of LAG-3 and its relationship to PD-1 will aid in patient selection and interpretation of clinical responses to combined inhibition. Methods: LAG-3 Ab clone EPR4392(2) (Abcam) and PD-1 Ab clone NAT105 (Ventana) were developed as individual or combined dual IHC assays on the Ventana Discovery Ultra platform. LAG-3 IHC was performed on panels of commercially available tissues: 33 normal human tissues (2 each) and various tumor types including anal (n = 39), cervical cancer (40), cholangiocarcinoma (11), DLBCL (40), gastric cancer (34), HCC (18), SCCHN (39), HER2-positive breast cancer (48), mesothelioma (40), NSCLC (39), ovarian cancer (47), SCLC (48), and TNBC (40). LAG-3 IHC expression was confirmed by in situ hybridization (ACD RNAScope). Positivity was defined as at least one LAG-3+ve or PD-1+ve tumor-infiltrating lymphocyte (TIL) per 40x magnification hot spot field (HSF); high LAG-3 expression was defined as > 15 LAG-3+ve TILs per HSF. Results: LAG-3-specific staining in normal tissues was limited to mononuclear cells in lymphoid organs and GALT, with no staining observed in normal parenchyma. Across various tumor types, the frequency of LAG-3+ve tumor samples and high expression, respectively (%, %), were as follows: cervical (100; 57.5), anal (97.4; 30.8), DLBCL (95; 75), NSCLC (92.3; 25.6), TNBC (90; 22.5), gastric (88.2; 44.1), SCCHN (87.2; 56.4), HER2-positive breast (85.4; 39.6), HCC (83.3; 22.2), cholangiocarcinoma (81.8; 9.1), ovarian (70.2; 21.3), SCLC (50; 14.6) and mesothelioma (25; 10). In addition to TILs, LAG-3 expression was observed on tumor cells in DLBCL and a fraction of anal, breast, cervical, gastric, hepatocellular, SCCHN, NSCLC and ovarian cancers. LAG-3/PD-1 double IHC staining revealed dual positivity across 92.3% of NSCLC samples, with approximately 60% demonstrating TIL co-expression of PD-1 and LAG-3. Conclusions: LAG-3 expression was detected on TILs across a broad range of solid tumors and DLBCL, with varying level of intensity or association with PD-1 expression. Correlative assessments of LAG-3/PD-1 expression with clinical responses to MGD013, an investigational bispecific DART(R) molecule targeting LAG-3/PD-1 (NCT03219268), will be undertaken.

CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner.

Great progress has been made in the field of tumor immunotherapy in the past decade. However, the therapeutic effects of immune checkpoint blockade (ICB) against ovarian cancer are still limited. Recently, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6i) has been reported to enhance antitumor immunity in preclinical models. The combined use of CDK4/6i and ICB may be beneficial, but the effects of CDK4/6is on the tumor immune microenvironment and whether they can synergize with ICB in treating ovarian cancer remain unknown.Methods: In this study, we first assessed the antitumor efficacy of abemaciclib, an FDA-approved CDK4/6i, in a syngeneic murine ovarian cancer model. Then, immunohistochemistry, immunofluorescence and flow cytometry were performed to evaluate the number, proportion, and activity of tumor-infiltrating lymphocytes. Cytokine and chemokine production was detected both in vivo and in vitro by PCR array analysis and cytokine antibody arrays. The treatment efficacy of combined abemaciclib and anti-PD-1 therapy was evaluated in vivo, and CD8+ and CD4+ T cell activities were analyzed using flow cytometry. Lastly, the requirement for both CD8+ T cells and B cells in combination treatment was evaluated in vivo, and potential cellular mechanisms were further analyzed by flow cytometry.Results: We observed that abemaciclib monotherapy could enhance immune infiltration, especially CD8+ T cell and B cell infiltration, in the ID8 murine ovarian cancer model. Immunophenotyping analysis showed that abemaciclib induced a proinflammatory immune response in the tumor microenvironment. PCR array analysis suggested the presence of a Th1-polarized cytokine profile in abemaciclib-treated ID8 tumors. In vitro studies showed that abemaciclib-treated ID8 cells secreted more CXCL10 and CXCL13, thus recruiting more lymphocytes than control groups. Combination treatment achieved better tumor control than monotherapy, and the activities of CD8+ and CD4+ T cells were further enhanced when compared with monotherapy. The synergistic antitumor effects of combined abemaciclib and anti-PD-1 therapy depended on both CD8+ T cells and B cells.Conclusion: These findings suggest that combined treatment with CDK4/6i and anti-PD-1 antibody could improve the efficacy of anti-PD-1 therapy and hold great promise for the treatment of poorly immune-infiltrated ovarian cancer.

Abstract 1559: TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models

Abstract Deficient anti-tumor immunity often results from an immunosuppressive tumor microenvironment (TME) that renders antigen presenting cells (APCs) unable to effectively stimulate T cells. Recent studies indicate that local delivery of immunostimulatory adjuvants can activate tumor resident APCs, driving uptake, processing and presentation of tumor neoantigens to T cells that mediate anti-tumor immunity. To overcome challenges associated with intratumoral delivery of such adjuvants, we developed a novel class of TLR immune-stimulating antibody conjugates (TAC) that comprise a TLR7/8 agonist conjugated to tumor-targeting monoclonal antibodies. In vitro co-cultures with human cancer cell lines and leukocytes revealed that TACs potently activate primary APCs, leading to increased co-stimulatory molecule expression (e.g. CD40, CD86) and secretion of pro-inflammatory cytokines (e.g. TNFα). The TACs also enhanced antibody-mediated effector functions such as ADCC and ADCP. Surprisingly, these constructs also induced dendritic cell (DC) differentiation from monocytes, as measured by changes in cellular morphology and DC surface markers (e.g. CD14 downregulation). CyTOF-based analysis of intracellular signaling in human leukocytes revealed a unique signaling signature of the conjugate compared to a mixture of its components, suggesting a novel biological mechanism by which the conjugate stimulates APCs. Finally, we demonstrated in vivo efficacy in syngeneic tumor models in which TAC treatment led to tumor clearance and development of immunologic memory. These results provide a strong rationale for this technology as a platform for cancer immunotherapy. Citation Format: Shelley E. Ackerman, Joseph C. Gonzalez, Josh D. Gregorio, Jason C. Paik, Felix J. Hartmann, Justin A. Kenkel, Arthur Lee, Angela Luo, Cecelia I. Pearson, Murray L. Nguyen, Benjamin Ackerman, Lauren Y. Sheu, Richard P. Laura, Steven J. Chapin, Brian S. Safina, Sean C. Bendall, David Dornan, Edgar G. Engleman, Michael N. Alonso. TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models [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 1559.

Abstract 4148: An orally bioavailable small molecule antagonist of TIM-3 signaling pathway shows potent anti-tumor activity

Abstract Activation of anti-tumor immune response by specific inhibition of PD1 pathway using monoclonal antibodies has now become of the mainstay in cancer therapy as evidenced by its widespread use in an expanding list of indications. Although these antibodies show impressive durable clinical activity, low response rates are witnessed in a large number of cancers, including colorectal cancer that remain largely refractory to PD-1 blockade. Upregulation of alternative immune checkpoints such as T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and VISTA contributes to the lack of response in patients not responding to therapies with anti-PD-1/PD-L1 antibodies. TIM-3 is a co-inhibitory receptor expressed on IFN-γ-producing T cells, FoxP3+ Treg cells and innate immune cells. Synergistic effects in restoring the anti-tumor immunity in preclinical models upon dual blockade of TIM-3 and PD-1 has provided a strong rationale for developing TIM-3 agents for use in combination with PD1 agents in the clinic. We sought to discover and develop an orally available small molecule antagonist targeting TIM3- signaling pathways. Unlike antibodies an oral agent potentially offers the convenience, flexibility to adjust dose and schedule to address any emergent adverse events and ease of combination therapy. Because TIM-3 shares sequence and structural similarity with the B7 family ligands, a focused library of compounds mimicking the interaction of checkpoint proteins of B7 family was screened towards the functional antagonism of TIM-3. Further optimization of the hit compounds resulted in lead compounds targeting TIM-3 pathway with desirable potency and selectivity. Lead compounds exhibited potent functional activity comparable to that obtained with an anti-TIM-3 antibody in rescuing the effector functions in human PBMC-based assays. Additionally, an advanced lead compound exhibited desirable drug-like properties including solubility, metabolic stability and pharmacokinetics with good oral bioavailability. In a syngeneic tumor models, once a day oral dosing of the advanced lead compound resulted in significant tumor growth inhibition as a single agent and in combination with anti-PD1 antibody that correlated well with immune PD in the tumor. The findings reported here support the development of the oral TIM-3 antagonist for use in the clinic. Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Raghuveer K. Ramachandra, Nagesh Gowda, Srinivaskumar Devarapalli, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Amit A. Dhudashiya, Dodheri S. Samiulla, Nagaraj M. Gowda, Murali Ramachandra. An orally bioavailable small molecule antagonist of TIM-3 signaling pathway shows potent anti-tumor activity [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 4148.

Abstract 3252: TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming

Abstract SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of the small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 is a novel, selective small molecule inhibitor of the SUMOylation enzymatic cascade, which has demonstrated induction of potent anti-tumor immunity in preclinical models. In this study, we evaluated changes in immune cell composition and states after TAK-981 treatment in preclinical models. In both mouse bone-marrow and human peripheral blood mononuclear cell derived dendritic cells (DCs), TAK-981 treatment ex vivo induced markers of activation and maturation including CD40, CD80 and CD86, as well as increased secretion of inflammatory cytokines like IP-10, MCP1, MIP-1α, MIP1β, IFNα and IFNβ. These effects were reversed by an interferon alpha receptor (IFNAR) blocking antibody, demonstrating dependence of TAK-981 induced pharmacodynamic effects on Type I interferon signaling in DCs. In vivo, a single sub-cutaneous injection of TAK-981 in naïve Balb/c mice at the brachial lymph nodes induced activation of DCs, measured as significant increases in CD40 and CD86 expression. Concomitantly, an increase in expression of the early lymphocyte activation marker CD69 was observed on T cells and NK cells, demonstrating pleiotropic effects of TAK-981 on immune cells. Increased expression of CD69 was also observed in purified human T and NK cells treated with TAK-981 ex vivo. To investigate if enhanced activation of DCs by TAK-981 leads to improved cross-presentation of exogenous antigens, we challenged naïve C57BL/6 mice with chicken-ovalbumin (OVA) protein alone or in combination with TAK-981. Interestingly, an increase in the frequency of Kb-SIINFEKL tetramer positive CD8 T cells was observed in the draining lymph nodes overnight, and in the spleen on day 14, after treatment with TAK-981+OVA, suggesting that TAK-981 promotes cross-presentation of SIINFEKL, the class-I MHC epitope of OVA, to cognate antigen-specific CD8 T cells. We also observed an increase in the frequency of CD8α+ DCs loaded with the peptide ‘SIINFEKL’ on H-2Kb (class-I MHC of C57BL/6 mice), providing direct evidence for enhanced antigen-cross presentation. Similar findings were observed with TAK-981 administered either sub-cutaneously or via the therapeutic route of intra-venous injection in the above OVA immunization model. These results suggest a mechanism by which TAK-981 may promote anti-tumor immune responses in mice via enhanced cross-presentation of exogenous antigens released by dying tumor cells, leading to priming and activation of antigen reactive cytotoxic T cells. Currently, TAK-981 is being evaluated in Phase 1 clinical trials in adult patients with metastatic solid tumors or lymphomas (ClinicalTrials.gov Identifier: NCT03648372). Citation Format: Mithun Khattar, Keli Song, Stephen Grossman, Kristina Xega, Xingyue He, Neeraja Idamakanti, Dennis Huszar. TAK-981: A first in class SUMO inhibitor in Phase 1 trials that promotes dendritic cell activation, antigen-presentation, and T cell priming [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 3252.

Abstract 1719: Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer

Abstract Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p<0.05). Combined therapy was superior to either monotherapy in the neoadjuvant setting (combo vs. anti-PD-1 and vs. anti-CTLA-4 p<0.05), whereas there were no significant differences between combined treatment and monotherapies in the adjuvant setting. Moreover, combined therapy in the neoadjuvant setting significantly prolonged survival compared to adjuvant combined treatment (p<0.05). Neoadjuvant combined therapy produced the most profound reduction in lung metastases when compared to monotherapy (combo vs. IgG, vs. anti-PD-1 and vs. anti-CTLA-4 p<0.01), or to adjuvant combined treatment. Neoadjuvant combined therapy was associated with the greatest amounts of SIINFEKL-tetramer+ TILs in resected primary tumors and also with increased CD8+ TIL density in both resected primary tumors (combo vs. IgG and vs. anti-PD-1 p<0.05) and secondary lung metastases (combo vs. IgG, vs. anti-PD1 and vs. anti-CTLA-4 p<0.05). Functional studies investigating the immune response of tumors treated with perioperative immunotherapies are ongoing. Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1719.

Single dose denileukin diftitox does not enhance vaccine-induced T cell responses or effectively deplete Tregs in advanced melanoma: immune monitoring and clinical results of a randomized phase II trial

BackgroundDepletion of CD25+ Tregs improves anti-tumor immunity in preclinical models. Denileukin diftitox is a recombinant fusion protein of human IL-2 and diptheria toxin fragment that also can kill CD25+ T...

Abstract LB-013: The immunomodulatory effect of JAK inhibitors enhances metastasis by impairing antitumor immunity in preclinical models of breast cancer

Abstract The JAK-STAT pathway is an attractive therapeutic target in breast cancer due to its frequent activation. Clinical trials testing JAK inhibitors in breast cancer are ongoing, making it important to understand the effect of this therapeutic approach on metastasis. While it is recognized that tumor growth in primary and metastatic sites is influenced by the local environment, little is known about the effect of targeted therapies on metastases or on host cells interacting with tumor cells at distant locations. Our goal was to determine the effect of JAK inhibitors on breast cancer in the bone environment, a common site of metastasis. Using patient biopsies and preclinical models of breast cancer metastasis, we demonstrate that the JAK-STAT pathway is active in bone metastasis, both in the cancer cells and in the tumor environment. To study the effect of JAK inhibitors, we established preclinical models of bone metastatic tumors, taking advantage of the bone tropism when injected via the intracardiac route, of the breast cancer cell lines, human MDA-MB231 scp1833 and mouse EO771. In vivo, in both models, STAT3 was active and treatment with the JAK1-JAK2 inhibitor ruxolitinib decreased pSTAT3 levels in primary tumors and in bone metastases. Unexpectedly, blocking the pathway with ruxolitinib, or with the JAK2 inhibitor BSK805, enhanced the metastatic burden in both models. To investigate the effect of JAK inhibition on tumor cell dissemination from the primary site, we employed the 4T1.2 model, which spontaneously metastasizes from the mammary tumor to the bone and the lungs. As seen with the other models, there was a significant increase in tumor cell numbers in the bone and in the lungs in response to JAK inhibitor treatment. To understand the mechanism underlying the increase in metastatic load, we considered the host immune system as a potential bystander target of the JAK inhibitors. Indeed, in response to JAK inhibitor treatment we observed a major reduction in the NK cell population. The effect of JAK inhibitors on NK cells was systemic since they were also reduced in the bone marrow and in the peripheral blood of tumor-bearing mice. To mechanistically explain the impact of JAK inhibitors on NK cells, we used the NK cell line, NK-92. Upon treatment of NK-92 cells with JAK inhibitors, activation of multiple STATs decreased and cell proliferation was strongly inhibited. In cytotoxic assays, treatment with JAK inhibitors significantly decreased the killing ability of NK-92 cells against carcinoma cells. To test the in vivo relevance of NK cells in metastatic growth we used NSG mice, which are devoid of NK cell activity. Remarkably, in contrast to the results obtained with 4T1.2 mammary tumors grown in immunocompetent hosts, no increase in bone or lung metastases was observed in tumor-bearing NSG hosts treated with ruxolitinib, providing strong evidence that JAK inhibition increases metastasis by interfering with NK cell mediated anti-tumor innate immunity. These results suggest that the immunomodulatory effect of JAK inhibitors in breast cancer patients undergoing clinical trials should be monitored. Moreover, our work highlights the importance of evaluating the effect of targeted therapy on cell populations in the tumor environment in order to predict and overcame bystander effects that might impact on therapy response. Citation Format: Nancy E. Hynes, Alessia Bottos, Jason Gill, Thomas Radimerski, Alexander Tzankov, Aleksandra Wodnar-Filipowicz. The immunomodulatory effect of JAK inhibitors enhances metastasis by impairing antitumor immunity in preclinical models of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-013. doi:10.1158/1538-7445.AM2015-LB-013