Tumor-free Survivors Research Articles

Abstract 4204: Subcutaneous vs. orthotopic tumor models: A comparative assessment

Abstract The failure of murine tumor models to adequately simulate the native biological milieu and tissue architecture of human malignancies, has fomented much discourse surrounding the discord that exists between experimental outcomes and clinical settings. Orthotopic (OT) models, which are generated by the engraftment of tumor cells or fragments into recipient organs of the same histotype from which they were derived, has the advantage of recapitulating the appropriate vasculature, as well as cellular and stromal components as the site of origin. In the context of immunotherapy, there is increasing evidence that subcutaneous (SC) tumors bear a vastly different immunological profile compared to OT implants, a phenomenon dictated by tissue-specific recruitment of various immune subsets, and subsequently, impacts overall response. Therefore, establishing cancer models that better represent the natural tumor environment is imperative for addressing some of the foibles inherent to preclinical mouse models. To demonstrate the significance of the anatomical site of the tumor, we present a comparative assessment of the MC38 murine colon carcinoma model, in the context of SC and OT settings. In the SC model of MC38, administration of either 5mg/kg anti-mPD1, anti-mPDL1, or anti-mCTLA-4, failed to elicit any notable anti-tumor activity, as evidenced by 0% incidences of complete regressions (CR) and tumor free survivors (TFS) for all three therapeutic agents, and incidences of 11%, 5% and -22% increase in time to progression (ITP), respectively. However, mice bearing OT tumors, exhibited meaningful response to 5mg/kg anti-mPD1 and anti-mPDL1, both evoking incidences of 25% CR, 25% TFS and 108% ITP. Though the anti-mCTLA-4 treated cohort did not produce any CRs or TFS, there was, nonetheless, a 50% incidence of ITP. Taken together, the data clearly illustrates the distinct disparities in response between SC and OT tumors, with respect to immunomodulatory agents. Characterization of immune phenotype within both settings, and interrogation of additional mouse models are ongoing. The exigencies and profound limitations associated with bench to bedside translation have long encumbered the drug development process. Despite staggering financial investment in oncology research, effective treatments for a myriad of cancers remain elusive, and only 3.4% of cancer drug candidates are conferred with clinical approval. Employing modalities that adequately reproduce the molecular and structural frameworks of human tumors, is crucial to better elucidating the natural trajectory of cancer progression, thus facilitating more accurate evaluation of drug activity and therapeutic response in preclinical settings. OT models therefore represent a more disease-relevant preclinical approach compared to their subcutaneous counterparts, as they more reliably preserve the intrinsic infrastructure and complexities of patient tumors in situ. Citation Format: Kerry-Ann Bright, Sheri Barnes, Derrik Germain, Lauren Kucharczyk. Subcutaneous vs. orthotopic tumor models: A comparative assessment [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 4204.

Abstract 3740: Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor

Abstract Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC) targeted to carcino-embryonic antigen-related cell adhesion molecule 5 (CEACAM5) and delivering DM4, a maytansinoid cytotoxic. CEACAM5 is highly expressed in several tumor types, including non-squamous non-small cell lung cancer (NSQ NSCLC). It is currently being evaluated in patients with advanced CEACAM5-positive NSQ NSCLC, in a phase II study in combination with pembrolizumab, and a phase III trial as single agent in comparison with docetaxel. Preclinical investigations were performed to determine whether SAR408701 could be beneficially combined with immune checkpoint inhibitors. We, first, demonstrated that the tusamitamab ravtansine payload, DM4, is able to induce robust immunogenic cell death (ICD) using an in vivo vaccination assay. In this assay, immunocompetent mice were “vaccinated” by DM4-treated CT26 cells and then later rechallenged with CT26 tumor cells. Tumor rejection was observed in more than 50% of “vaccinated” mice. The ICD was characterized by the expression or release of danger-associated molecular patterns (DAMPs), and we observed DAMP modulation such as calreticulin (CRT) exposure at the cell surface of dying tumor cells, and the release of the High-Mobility Group Box 1 (HMGB1) in the extracellular space by CT26 cells in response to DM4 treatment. We also showed that DM4-treated CT26 tumor cells implanted into mice or SAR408701 treatment of MC38 tumor-bearing mice induced an elevation of plasmatic level of murine IL-6 and INFγ, revealing potential antitumor immunity modulation by the ADC and its payload. Immunohistochemistry analysis of MC38 tumors also revealed that tusamitamab ravtansine treatment induced a dose-dependent increase of CD8 T-cell infiltration in tumors correlating to dose-dependent efficacy. Finally, tusamitamab ravtansine was combined with immune checkpoint inhibitors (ICIs) in immunocompetent mice bearing MC38 tumor model. In this model, the combination of tusamitamab ravtansine with anti-PD-1 or anti-PD-L1 antibody led to complete response and, even, tumor-free survivors at 120 days post tumor implantation while single agents were inactive or transiently active. In summary, these preclinical data show that tusamitamab ravtansine could be beneficially combined with immune checkpoint and strongly support their evaluation in clinical development. Citation Format: Celine Nicolazzi, Anne-Marie Lefebvre, Nicolas Moindrot, Christelle Larois, Marion Classe, Sukhvinder Sidhu. Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor [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 3740.

Abstract 1604: Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2

Abstract Interleukin 13 receptor subunit alpha 2 (IL13RA2) is one of the two major receptors for the cytokine interleukin 13 (IL-13). While IL13RA1 has low affinity for IL-13 and it is expressed ubiquitously in humans, IL13RA2 has high binding affinity to IL-13 and its expression in normal tissues is mainly restricted to the testes. IL13RA2 was initially regarded as a decoy receptor because it has a short cytoplasmic tail with no known signaling motifs, and its binding to IL-13 does not lead to activation of the JAK/STAT6 pathway. However, recent studies demonstrated that IL-13-mediated IL13RA2 signaling occurs via STAT6-independent pathways. IL13RA2 is reported to be expressed at a high frequency in glioblastoma multiforme (GBM) as well as in other solid tumors including melanoma, renal cell carcinoma and adrenocortical carcinoma and is correlated with poor prognosis. ADCT-211 is an antibody-drug conjugate composed of HuCl47, a humanized IgG1 antibody directed against human IL13RA2, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG2000 (drug to antibody ratio ~ 1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-211 in human cancer cell lines and xenograft models, to determine its safety, tolerability and pharmacokinetic (PK) in the rat and to measure IL13RA2 expression by immunohistochemistry (IHC) in human tumor specimens. HuCl47 showed specific binding to recombinant IL13RA2, while it did not bind to IL13RA1. In vitro, ADCT-211 had highly potent and targeted cytotoxicity against various IL13RA2-expressing solid cancer cell lines. In vivo, a single dose of ADCT-211 at 10 mg/kg was able to completely eradicate tumors in the subcutaneous (sc) A375 human melanoma xenograft model and resulted in 8/8 tumor-free survivors (TFS) at the end of the study on day 57. In the sc U251 human glioblastoma xenograft model, a single dose of ADCT-211 at 3.75 mg/kg showed potent and durable antitumor activity and it resulted in 5/10 partial responders and 5/10 complete responders, 2 of which were TFS at the end of the study on day 51. In an MTD study in male rats, ADCT-211 was stable and tolerated up to 24 mg/kg single dose, with exposure data being indicative of a linear PK profile with a half-life of 12-19 days. Expression of membranous IL13RA2 was confirmed by IHC in a panel of primary and refractory GBM samples and malignant melanoma, highlighting them as potential indications for the clinical development of ADCT-211. In conclusion, ADCT-211 demonstrated potent and specific in vitro and in vivo anti-tumor activity and it was stable and well tolerated in the rat, warranting further development of ADCT-211 into the clinic in IL13RA2-expressing cancers. Citation Format: Francesca Zammarchi, Karin Havenith, Ben Leatherdale, Britanny Roberts, Lara Montolio, Afroze Patel, Narinder Janghra, Pedro Alves, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Lolke de Haan, Patrick H van Berkel. Preclinical development of ADCT-211, a novel pyrrolobenzodiazepine dimer-based antibody-drug conjugate targeting solid tumors expressing IL13RA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1604.

Abstract 2280: BG34-200 immunotherapy of advanced melanoma

Abstract Background: High levels of myeloid derived cells are characteristic of tumor microenvironment (TME) of advanced melanoma. These cells work with the tumor cells to suppress host development of anti-tumor immune responses, regulate tumor metastasis, and drive cancer drug resistance to virtually all types of therapy. Therefore, methods to disrupt tumor-associated myeloid cell function are actively being sought to find a cure. Goals: Our team has recently developed a plant-derived carbohydrate molecule, BG34-200, that modulates tumor-associated myeloid cells by targeting the cell surface receptor CD11b. In this study, we investigated the immune activation induced by BG34-200-CD11b engagement in the context of advanced melanoma and use the knowledge to develop BG34-200-based combination immunotherapy. Methods: Wild type B57Bl6/J mice were injected with 1.5million B16F10 cells and treated intravenously with BG34-200. Tumor volume and animals survival were monitored, and whole blood was drawn for FACS analysis. Results: We found that BG34-200 IV administration could significantly inhibit tumor growth and improve survival in B16F10 mice with advanced melanoma. Our data supported a model that the entry of BG34-200 into circulating melanoma tumor-associated inflammatory monocytes (TAIMs) could trigger a sequential immune activation: the BG34-200+ TAIM subsets migrated to tumor and differentiated into monocyte-derived dendritic cells (mo-DCs); then, the BG34-200+ mo-DCs migrated to tumor draining lymph nodes where they triggered the generation of tumor antigen specific T cells. Based upon these results, we combined BG34-200 treatment with adoptive transfer of TdLN-derived T-cell to treat advanced melanoma, which significantly improved animal survival and helped tumor-free survivors be resistant to a second tumor cell challenge. Conclusions: The scientific findings from this study will allow us to develop new technology to apply BG34-200-based immunotherapy to patients with advanced melanoma who have not responded to current standard of care therapies with and without immunotherapy. Citation Format: Veronique P. Roche, Victor Sandoval, Zachary Senders, Joshua Lyons, Claire Wolford, Mei Zhang. BG34-200 immunotherapy of advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2280.

Abstract 3198: Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo

Abstract Introduction: Despite significant progress in the chimeric antigen receptor (CAR)-T cell therapy of B cell malignancies, the 5-year progression-free survival rate in large B cell lymphoma following CD19-targeting CAR-T cell treatments remains below 50%. While some treatment failures were attributed to CD19 antigen loss, immunogenicity of murine single-chain fragment variable (scFv) elements has been implicated in relapse and resistance to CAR-T cell re-treatment. In this study, we evaluated fully human CD20 and CD19 dual-targeting CAR constructs (h20.19 CARs) combining human scFvs and distinct co-stimulatory domains and architectures, to mitigate tumor antigen escape, reduce immunogenicity, and improve CAR functionality. Methods: The h20.19 CAR constructs were assembled from fully human scFvs against lymphoma antigens CD19 and CD20, with hinge, transmembrane and co-stimulatory domains derived from 4-1BB, CD28, OX40 or ICOS. Tandem second generation (T8/8BBz), tandem third generation (T28/28BBz, T8/28BBz), and bicistronic (D28z/BBz, DBBz/28z, DOXz/ICz) CARs were evaluated. CAR T cells were derived from healthy donors’ CD4+ and CD8+ T cells by lentiviral transduction, and CAR expression was evaluated by flow cytometry. CAR-mediated target cell killing was assessed by co-culture assays, and cytokine elaboration by ELISA. In vivo CAR-T cell function was evaluated in a Raji lymphoma NSG xenograft model using low CAR-T dose (2x106/mouse). Tumor-free survivors were rechallenged with Raji cells to compare CAR-T cell potency and persistence. Results: The hCD20.19 CAR-T cells lysed CD19+CD20+ tumor lines Raji, NALM-6 and REH, and single-antigen lines 293T CD19 and 293T CD20, but not the parental CD19-CD20- 293T cells, indicating antigen-specific efficacy. Cytokines TNFα, IL2 and IFNγ were strongly upregulated in response to Raji cells. During repeat co-culture with Raji cells, the third-generation tandem hCD20.19 CARs and the bicistronic CARs showed greater cytotoxicity than the second-generation CAR T8/8BBz. In vivo, all third-generation tandem and bicistronic CARs induced rapid and durable Raji tumor rejection by study day 7 post CAR-T, with no apparent toxicity. Upon long rest and rechallenge with Raji cells on study day 50, all h20.19 CARs attenuated tumor progression as compared to age-matched untreated mice cohort. CARs T8/28BBz and D28z/BBz afforded the least protection from Raji rechallenge, whereas CAR DOXz/ICz mediated rapid tumor rejection, but mice met euthanasia criteria by study day 71 due to weight loss. The tandem CAR T28/28BBz and bicistronic CAR DBBz/28z, demonstrated tumor rejection, persistence and safety after rechallenge, up to study day 86. Conclusions: In summary, the fully human dual-targeting h20.19 CARs exhibit robust anti-tumor function and persistence in vitro and in vivo, mediate long-term remissions and resist tumor rechallenge. Citation Format: Peirong Hu, Brittany Steimle, Ngoc Tran, Pradyot Dash, Dina Schneider. Fully human CD20 and CD19 dual-targeting CAR-T cells mediate long-term remissions and protection from tumor rechallenge in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3198.

Abstract OT3-28-01: A Dose-escalation Study of the Safety and Pharmacology of DAN-222 in Subjects with Metastatic Breast Cancer NCT05261269

Abstract Clinical Study: This clinical study is designed to evaluate DAN-222 as a monotherapy and in combination with a PARP inhibitor which is expected to increase efficacy without significant increase in toxicity. DAN-222 is a novel therapeutic nanoparticle with a complimentary mechanism for combination with a PARP inhibitor anticipated for patients with HRD+ and HRD- tumors, and not restricted to BRCAm. This is an ongoing phase 1/2 study to evaluate the safety and pharmacology of DAN-222 in patients with metastatic breast cancer and initial evaluation of efficacy. Here we report on the initial pharmacology. The results in the patients show the expected characteristics of the designed product, including consistency with preclinical models (e.g. T1/2= 28 hours), which is a feature of this platform given the non-enzymatic release mechanism of the payload that allows for improved translation from preclinical species to clinical patients as well low variability between patients (e.g. CV%=16.5). Study Rationale: DAN-222 is a topoisomerase-1 inhibitor (Camptothecin) nanoparticle therapeutic that has been optimized for tumor biodistribution and pharmacokinetics. DAN-222 has a broad therapeutic index in preclinical evaluation and the complementary mechanism of action with PARP inhibitors provides significantly enhanced efficacy while also sparing bone marrow. Importantly, the complementary enhanced efficacy is independent of tumor homologous repair deficiency (HRD) status, including BRCA status. The efficacy of DAN-222 was evaluated alone and in combination with a PARP inhibitor (niraparib) in HRD+ breast cancer (MDA-MB-436) and HRD- ovarian cancer (OVCAR-3) xenograft models. Table 1 highlights the endpoints of the study as measured by partial response, complete response, tumor free survival and median tumor volume at end of study (Day 60). DAN-222 alone had PR effects and reduced median tumor volume compared to niraparib alone. The combination demonstrated enhancement of response as evidenced by an increase in partial response, a shift from partial to complete response, an increase in tumor-free survivors, and significant further reduction in median tumor volume. Conclusion: The clinical pharmacology will be presented and demonstrates the designed behavior of the nanoparticle. A design feature of the nanoparticle is that the payload and linker are sequestered in the core, protecting them from circulatory components. Moreover, the covalent attachment of the payload allows for tunable release kinetics via a hydrolytic linker, preventing burst release and associated toxicities common to physical encapsulation-based nanoparticle systems (e.g., liposomes, micelles). The preliminary PK profile in patients supports the translatability across species and consistency of exposure across patients. Table 1: Efficacy of DAN-222 Alone or Combination with Niraparib Citation Format: Ashley P. Wright, Emily A. Wyatt, Timothy Hagerty. A Dose-escalation Study of the Safety and Pharmacology of DAN-222 in Subjects with Metastatic Breast Cancer NCT05261269 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-28-01.

BG34-200 Immunotherapy of Advanced Melanoma.

High levels of myeloid-derived cells are characteristic of the tumor microenvironment (TME) of advanced melanoma. These cells interact with tumor cells to suppress the development of antitumor immune responses, regulate tumor metastasis, and drive cancer's resistance to virtually all types of therapy. Therefore, methods to disrupt tumor-associated myeloid cell function are actively being sought to find a cure. Our team has recently developed a plant-derived carbohydrate molecule, BG34-200, that modulates tumor-associated myeloid cells by targeting the cell surface receptor CD11b. In this study, we found that BG34-200 IV administration could significantly inhibit tumor growth and improve survival in B16F10 mice with advanced melanoma. Our data supported a model that the entry of BG34-200 into circulating melanoma tumor-associated inflammatory monocytes (TAIMs) could trigger a sequential immune activation: the BG34-200+ TAIM subsets migrated to tumor and differentiated into monocyte-derived dendritic cells (mo-DCs); then, the BG34-200+ mo-DCs migrated to tumor draining lymph nodes, where they triggered the generation of tumor-antigen-specific T cells. Based upon these results, we combined BG34-200 treatment with adoptive transfer of TdLN-derived T cells to treat advanced melanoma, which significantly improved animal survival and helped tumor-free survivors be resistant to a second tumor-cell challenge. The scientific findings from this study will allow us to develop new technology and apply BG34-200-based immunotherapy to patients with advanced melanoma who have not responded to current standard of care therapies with and without immunotherapy.

Survival rate and quality of life of human papillomaviruse-negative patients with advanced oropharyngeal cancer receiving different treatments

To analyze the survival rates and quality of life of human papillomaviruse (HPV)-negative patients with advanced oropharyngeal cancer after different combined treatments with chemotherapy, surgery and radiotherapy. We retrospectively analyzed the data of patients with oropharyngeal cancer hospitalized in our hospital from January, 2015 to December, 2020, and after case analysis of the clinical, imaging and pathological data, 405 patients were included in this study and grouped according to the treatments they received. Kaplan-Meier and Log-rank analysis were used to calculate the overall survival rate and the survival rate of patients with different treatments. The self-rated quality of life of the tumor-free survivors was assessed using UW-QOL (4) questionnaire and compared among the patients with different treatments. Among the 405 patients included in this study, 146 received treatments with chemotherapy+surgery+radiotherapy (CSRT), 138 received surgery+radiotherapy (SRT) and 121 were treated with radiotherapy+chemotherapy (RCT). The overall survival rates of the 3 groups at 1, 3 and 5 years were 85.1%, 67.1% and 56.9%, respectively, and the survival rates of patients receiving CSRT, SRT and RCT did not differ significantly (P > 0.05). A total of 280 UW-QOL (4) questionnaires were distributed and 202 (72.14%) were retrieved. The average total scores decreased in the order of CSRT > SRT > RCT; the scores were significantly higher in CSRT group than in SRT and RCT (P < 0.05), but did not differ significantly between SRT and RCT groups (P > 0.05). CSRT, SRT and RCT are all treatment options for locally advanced oropharyngeal cancer, but CSRT may achieve better quality of life of the patients than SRT and RCT.

Abstract 1617: Systematic evaluation of Cloudman S91 and YUMM1.7 murine melanoma models to immunotherapy and focal radiation

Abstract In the preclinical setting, the B16-F10 syngeneic melanoma model is commonly used but is highly refractory to most immunomodulators, limiting the applicability of this model. To expand our offerings of melanoma models beyond B16-F10, we have characterized two syngeneic melanoma models - Cloudman S91 and YUMM1.7, for preclinical oncology studies. Our in vivo (immune-oncology and radiation) and in vitro (flow cytometry) analyses establishes that Cloudman S91 is a more responsive model compared to YUMM1.7. The two models were evaluated for their responses to immune modulatory agents and focal radiation. Single agent treatment of established Cloudman S91 tumors with checkpoint inhibitors (CPI) anti-mPD-1 and anti-mPD-L1 resulted in 116% and 41% increase in time to progression (%ITP) respectively. Treatment with anti-mCTLA-4, on the other hand, resulted in over 283% ITP with 20% tumor free survivors (TFS). Contrastingly, YUMM1.7 had no response to checkpoint blockade. These results indicate Cloudman S91 as a more responsive model to checkpoint blockade compared to YUMM1.7. Both models were found to be sensitive to focal beam radiation (RT) delivered using the Small Animal Radiation Research Platform (SARRP; Xstrahl) resulting in dose dependent tumor growth delay in both models. In order to determine if the addition of CPI to RT could improve the outcome, we tested anti-mPD1 in combination with RT at 5Gy. Both models had improved response to combination treatment, with YUMM1.7 showing a 43% ITP and the Cloudman with &amp;gt;350% ITP over untreated controls and 15% and 170% over RT single treatment. Analysis of untreated tumors by flow cytometry shows that both tumor models have a large myeloid population and relatively low lymphoid infiltrate. Changes in immune profiles upon treatment with anti-mPD1 and RT or in combination were further analyzed. Notably, RT treatment alone increased CD8+ T cell infiltration and decreased the M2 macrophage infiltration to the tumors resulting in a less immunosuppressive tumor microenvironment. In the combination groups, this microenvironment enhances the response to immunotherapy as seen in the improvement in activity compared to RT alone. Here we demonstrate the varying responses within the same histotype to similar treatments to help in drug discovery programs. Combination of radiotherapy with immunotherapy offers benefits of modulating immune cells and enhancing the local and potentially abscopal effects of immunotherapy in both the Cloudman S91 and YUMM1.7 melanoma models. These characterizations can be useful in guiding model selection of murine melanoma and can inform rational combination treatment approaches for data driving decisions in drug development. Citation Format: Sumithra Urs, Derrik Germain, Olivia Nelson, Lauren Kucharczyk, David Draper, Sheri Barnes. Systematic evaluation of Cloudman S91 and YUMM1.7 murine melanoma models to immunotherapy and focal radiation [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 1617.

Abstract 2921: Characterization of orthotopically implanted syngeneic cell lines as more clinically relevant in vivo models

Abstract Preclinical oncology studies aim to model human disease in animal settings. To this effect, the site of tumor implant is thought to impact critical parameters that determine response to treatment. The more frequently used subcutaneous (SC) models have advantages; speed, cost efficiency and ability to monitor growth by caliper measurements but they lack heterogeneous tumor morphology, vessel density and immune cell infiltrates comparable to human disease. Orthotopic (OT) tumor models may allow for tumor modeling in the organ of origin by mimicking many human disease parameters such as vascularization, tumor architecture, metastatic potential, immune profile and tumor microenvironment. OT models may better represent clinical disease but are more challenging to monitor in vivo. Thus, we stably transfected several mouse syngeneic cell lines with luciferase (Luc). The Luc enabled cells facilitate non-invasive monitoring of tumor progression, potential metastasis and response to treatment via bioluminescence imaging (BLI). Here, we focus on pancreas (Pan02-Luc) and lung (LL/2-Luc) models. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Surgical procedures were used with minimal invasion to the respective organs followed by monitoring of tumor take and progression using BLI. Both the LL/2-Luc and Pan02-Luc models showed 90-100% tumor take and successful growth in their respective organs. The OT LL/2-Luc model showed mild response to paclitaxel and cisplatin with an increased time to progression (ITP) of 23% and 46%, respectively, 30% and 46% ITP to checkpoint inhibitors anti-PD-1 and anti-PD-L1 respectively, and 20% ITP to focal radiation (Xstrahl). In comparison, the SC LL/2 model showed 5%, 10%, 4.5% and 1% ITP to cisplatin, paclitaxel, anti-PD-1 and anti-PD-L1, respectively. This demonstrates the potential for the OT model to be more translatable than the SC model. Immune profile of the OT tumors by flow cytometry showed a high percentage of B cells and macrophages in the LL/2-Luc tumors. The OT Pan02-Luc model responded well to gemcitabine treatment with &amp;gt;96% ITP and 62% tumor free survivors while anti-PD-1 treatment resulted in 25% ITP. The SC Pan02 model showed 85% and 9% ITP to gemcitabine and anti-PD1 treatments, respectively. The baseline immune profile of Pan02-Luc tumors showed very few CD4+ and CD8+ T cells and a large myeloid cell population of M-MDSC &amp; M2 TAMs. The poor lymphoid cell infiltration and presence of large numbers of myeloid suppressor cells, characteristic of non-immunogenic tumors corresponds to the lack of robust response to checkpoint inhibitor treatment. The use of OT models may provide an important bridge between the more standard SC models and further clinical approaches. Citation Format: Sumithra Urs, Derrik Germain, Meridith Baugher, Erin Trachet, Maryland Franklin. Characterization of orthotopically implanted syngeneic cell lines as more clinically relevant in vivo models [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 2921.

Abstract 2926: CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response

Abstract Colorectal cancer (CRC) is a major contributor to the whole of cancer as a disease, representing 10% of all cases of cancer. Recent clinical studies and FDA approvals demonstrate strong possibilities for the future of immunotherapy in the treatment of CRC. Highly translational preclinical models of the disease are required to assess the effectiveness of novel immuno-oncology agents. The CT26.WT murine colon carcinoma model is a widely used syngeneic tumor model to evaluate the preclinical efficacy of immuno-oncology agents. Subcutaneous tumor models are limited in translatability compared to the orthotopic implant site including differential stromal interactions, immune cell infiltrate, and response to immunotherapy. We present the development of a robust murine orthotopic colon tumor model (CT26.WT-luc) assessed by bioluminescence imaging (BLI) and assessed for response to common checkpoint targets. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Subcutaneous CT26.WT-luc tumors were dissected into fragments and grafted onto the cecum of naïve Balb/c mice. Mice were subjected to BLI twice weekly for four weeks for longitudinal monitoring of tumor growth. The tumor engraftment rate was 95% based on BLI and necropsy, no spontaneous regressions were observed. The BLI derived tumor volume doubling time was 1.5 days and the median time on study was 21 days. Common clinical observations associated with disease progression were abdominal distension due to tumor growth and bodyweight gain. Necropsy revealed large primary masses attached to the cecum with large vascular involvement, and small nodules on the liver and abdominal wall. To assess the model response to immunotherapy, Balb/c mice bearing orthotopic CT26.WT-luc tumors were treated by intraperitoneal administration of 10mg/kg isotype control, anti-PD-L1, anti-CTLA-4 or anti-PD-1 twice weekly for two weeks (Bio X Cell). Administration of anti-PD-L1 resulted in one complete regression. Anti-CTLA-4 treatment resulted in a day 23 ΔT/ΔC of 0.1% and five partial regressions with no visibly detectable disease at necropsy. Administration of anti-PD-1 did not result in regressions or tumor free survivors. Administration of either anti-PD-L1 or anti-CTLA-4 provided an increase of life span over isotype control of 13 and 24 days, respectively. Terminal necropsy revealed no evidence of tumors or other lesions in 1 out of 8 animals administered anti-PD-L1, and 5 out of 8 animals administered anti-CTLA-4. This model represents a powerful tool with translational potential for assessing the efficacy of novel immune modulating agents with the possibility of combination therapy. Assessment of tumor response, cell and molecular differences between subcutaneous and orthotopic CT26.WT-luc tumors is ongoing. Citation Format: Patrick Allison, Derrik Germain, Meredith Baugher, Maryland Franklin. CT26.WT-luc orthotopic syngeneic tumor model of colon cancer: development and immunotherapy response [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 2926.

Abstract 1562: Syngeneic MC38 mouse colorectal tumor model: Immune profile and response to immunomodulatory agents and radiation

Abstract With 53,200 deaths expected in 2020, colorectal cancer (CRC) is the second most common cause of cancer deaths in the US. Metastatic lesions are common, and CRC remains incurable for many patients, leading to over 900,000 deaths per year worldwide. There is a need to develop safe and effective treatments since not all patients or tumors respond to current therapies and the use of appropriate animal models remains vital. One such model is the MC38 murine CRC cell line, derived from a grade-III adenocarcinoma chemically induced in a female C57BL/6 mouse. The aim of this study was to analyze the immune infiltration in subcutaneous MC38 tumors and track responses to various therapeutics. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. As shown by flow cytometry, naïve tumors in the log phase of growth exhibited low infiltration of CD45+ cells. Infiltration of lymphoid cells, represented by CD8+ T cells and CD4+ T helper cells, was also minimal, characteristic of a cold immune-suppressive landscape. The myeloid population represented ~50% of the CD45+ cells and was characterized predominantly by M1-TAMs, M-MDSCs, M2-TAMs and G-MDSCs, also implicated in immune-suppressive environments. To evaluate the effect of immunotherapies, mice bearing established tumors were administered various checkpoint inhibitors or costimulatory antibodies. The costimulatory antibodies anti-GITR, anti-CD137 and anti-OX40 showed no evident anti-tumor effect. In contrast, anti-PD-L1 and anti-PD-1 treatments produced clear anti-tumor activities with 40 and 50% putative responders and 35 and 37% increase in time to progression, respectively, as compared to controls. Tumor volumes in anti-PD-L1 and anti-PD-1 treated mice were also smaller than those in isotype controls starting day 22 post-tumor inoculation (509+103 and 504+94mm3 vs 899+124mm3, respectively, on day 22). Because radiotherapy is thought to increase the immunogenicity of tumors, we investigated the effects of focal radiation (RT) treatment (10Gy), delivered by the Small Animal Radiation Research Platform (Xstrahl), alone or in combination with checkpoint inhibition. Treatment with RT alone resulted in tumor growth delay of 12 days and an increase in time to progression of 119 %, as compared to controls, and 20% partial regression. Treatment with RT combined with anti-PD-1 potentiated these effects, with tumor growth delay of 14 days, an increase in time to progression of 134%, 30% complete regression, 10% partial regression, and 30% tumor free survivors. These results demonstrate that RT can convert an immune-suppressive milieu as seen in the MC38 model, into a warmer environment, and underscore the value of such relevant mouse models in developing clinically relevant therapeutic multimodality strategies. Citation Format: Sylvie Kossodo, Sheri Barnes, David Draper, Derrick Germain, Scott Wise, Maryland Rosenfeld Franklin. Syngeneic MC38 mouse colorectal tumor model: Immune profile and response to immunomodulatory agents and radiation [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 1562.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Abstract 6650: Targeted radiation in combination with immunotherapy agents in murine breast cancer models

Abstract Clinically, most breast cancers are refractory to checkpoint inhibitor (CPI) monotherapy and are regarded as immunologically “cold” due to minimal tumor CD8+ T cell infiltrate and low immunogenicity. These characteristics have encouraged the evaluation of potential synergy of immunotherapy with other treatment modalities such as targeted radiotherapy (RT). While RT by itself can have many immunostimulatory effects, it is insufficient to induce a curative anti-tumor immune response especially against metastatic breast cancers, highlighting the need for combination strategies that boost the immune system. Here, we evaluate three syngeneic triple negative breast cancer models, 4T1, E0771 and EMT6 and their responses to immune modulatory agents and focal radiation. Single agent CPI and costimulatory antibodies show anti-tumor activity with tumor growth delay (TGD) (varying from 1-24 days) and tumor free survivors (TFS) in the EMT6 model, whereas E0771 and 4T1 show little/no response to CPI treatment. All three models are sensitive to focal beam radiation (5-20Gy) delivered using the Small Animal Radiation Research Platform (SARRP; Xstrahl). RT monotherapy results in TGD in all three models. Combination of immune modulators and RT were evaluated in these models. The EMT6 model is the most responsive with RT in combination with anti-mPD-1 resulting in 9 days TGD (anti-mPD-1 monotherapy = 1.2 TGD) and 33 days TGD in combination with anti-mCD137 (anti-mCD137 monotherapy = 24 TGD) along with 50% TFS. RT in combination with anti-mCTLA-4 in the 4T1 model improves overall treatment response. The baseline tumor immune profile for the three models was established to understand their potential immunogenicity. The T and B cell populations (relative to CD45+) was comparable between models. In the myeloid compartment, 4T1 and EMT6 models are similar with respect to G-MDSCs (Granulocytic Myeloid Derived Suppressor Cells) being the predominant subset. On the other hand, E0771 tumors have a predominating M-MDSC (Monocytic-MDSC) population and a near absence of G-MDSCs. MDSCs are well known for contributing to immunosuppression and can play a role in promoting tumor metastasis by participating in the formation of angiogenesis and invasion. We determine that in the metastatic 4T1 model, combination treatment of anti-mCTLA-4 with RT reduces overall metastatic tumor burden in the lung and axillary lymph nodes which was concomitant with reduced MDSCs in the primary tumor. We have luciferase-enabled these cell lines to further evaluate combination therapy on metastatic spread. Combination of radiotherapy with immunotherapy offers benefits of targeting the tumor, activating immune cells and enhancing the local and potentially abscopal effects of immunotherapy. These data can be useful in providing a guide to choose between three murine breast cancer models for rational design of combination strategies. Citation Format: Sumithra Urs, David Draper, Maryland R. Franklin. Targeted radiation in combination with immunotherapy agents in murine breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6650.

Abstract 2257: Targeted thorium-227 conjugates demonstrate synergistic activity in combination with PD-L1 inhibitors

Abstract Targeted thorium-227 conjugates (TTCs) represent a new class of targeted alpha therapy (TAT). TTCs consist of an antigen targeting moiety, which is covalently attached to a 3,2-HOPO chelator, enabling specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. TTCs have demonstrated potent preclinical in vivo activity in monotherapy and in combination with DNA damage inhibitors. In the present work, the immunostimulatory effects of the mesothelin (MSLN) targeted thorium-227 conjugate (MSLN-TTC) was studied in immunocompetent mice, bearing human MSLN-transfected MC38 cells, in monotherapy and in combination with the PD-L1 inhibitor (PD-L1i) atezolizumab. Surface expression of DAMPs (calreticulin, HMGB1, HSP70 and HSP90), immunoinhibitory markers (PD-L1 and CTLA-4) and immunostimulatory markers (IFNAR, B7H2 and ICOS-L) was analyzed on murine, human MSLN-transfected MC38 colorectal cancer cells (MC38-hMSLN cells) after a 3-day exposure to MSLN-TTC. In vivo, C57/Bl6 mice were inoculated s.c. with MC38-hMSLN cells and treated with a single i.v. dose of non-radiolabeled MSLN-conjugate (0.14 mg/kg), isotype control-TTC (250 kBq/kg; 0.14 mg/kg), MSLN-TTC (250 kBq/kg; 0.14 mg/kg), PD-L1i (1.5 mg/kg; Q3D4; i.p.) or MSLN-TTC/PD-L1i combination. Tumors, lymph nodes, spleen and plasma were analyzed for MSLN and molecular markers. Tumor-free survivors were re-challenged with MC38-hMSLN or vector cells 125 d post-treatment. In a parallel arm of the experiment, CD8+ T cells were depleted using YTS169.4 antibody. MSLN-TTC treatment resulted in a specific dose-dependent increase of IFNAR, PD-L1 and HMGB1 in MC38-hMSLN cells in vitro. MSLN-TTC (T/C 0.38; Day 22) and PD-L1i (T/C 0.0.42; Day 22) monotherapies showed specific antitumor activity (T/C of radiolabeled isotype control was 0.76, Day 22) and their combination was synergistic (T/C 0.08; Day 22) with increased numbers of tumor free survivors. All three treatments increased HMGB1 levels in plasma. Induction of DSBs was observed in MSLN-TTC (+/- PD-L1i) treated tumors. Re-challenge of tumor-free survivors treated with MSLN-TTC, PD-L1i or MSLN-TTC/PD-L1i resulted in rejection of MC38-hMSLN, but not vector-cells. Infiltration of CD8+ T cells into tumors and their increase in tumor-draining lymph nodes were observed in MSLN-TTC, PD-L1i and MSLN-TTC/PD-L1i-treated groups but not in the vehicle group. Depletion of CD8+ T cells decreased the efficacy of PD-L1i and to a lower extent, MSLN-TTC and MSLN-TTC/PD-L1i. These results suggest that the efficacy of MSLN-TTC may involve activation of CD8+ T cells. These findings support the hypothesis that upon binding to antigen-positive cancer cells, TTC treatment induces DNA double strand breaks, leading to apoptosis and potentially immunogenic cell death, resulting in activation of the adaptive immune system. Citation Format: Urs B. Hagemann, Pascale Lejeune, Lisa Bartnitzky, Kathleen Stadelmann, Veronique Cruciani, Roger M. Bjerke, Sandra Berndt, Claudia Kamfenkel, Manuela Brand, Manuela Steinbach, Stefan Stargard, Christoph A. Schatz, Jenny Karlsson, Hartwig Hennekes, Alan S. Cuthbertson, Dominik Mumberg. Targeted thorium-227 conjugates demonstrate synergistic activity in combination with PD-L1 inhibitors [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 2257.

Abstract 5179: Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia

Abstract CD38 is highly expressed in many hematological malignancies such as multiple myeloma (MM) and acute lymphocytic leukemia (ALL) and can be targeted by antibody-based therapy. Isatuximab, an anti-CD38 IgG1 monoclonal antibody has promising clinical activity in relapsed/refractory multiple myeloma patients. It induces tumor cells death via Fc-mediated cytotoxic effects, direct cancer cell killing, and inhibition of CD38 enzymatic activity. We investigated the mechanisms of action (MoA) of isatuximab against MM and ALL in vitro and evaluated its in vivo pharmacological potency in combination with standard therapies for these two hematologic malignancies. Isatuximab preferentially induced robust antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) in both MM and ALL cell lines. Cancer cells with particularly high level of CD38 were also susceptible to apoptosis or CDC induced by isatuximab. In vitro, isatuximab displayed time- and dose-dependent inhibitory effect on CD38 enzymatic activity shown in LP-1 myeloma cells, suggesting that it could alleviate immunosuppressive effects of CD38-mediated adenosine synthesis in the tumor microenvironment. In vivo efficacy studies were conducted in mouse xenograft models. The in vivo efficacy of isatuximab in combination with the immunomodulatory drugs (IMiDs) pomalidomide or lenalidomide that are standard of care in MM was evaluated. Each combination increased the inhibition of tumor growth (T/C) compared with isatuximab or IMiDs treatment alone. Combination of isatuximab with either bortezomib or carfilzomib, two proteasome inhibitors that are widely used in MM, significantly improved their anti-tumor activity versus each agent alone. Isatuximab also improved tumor growth inhibition mediated by alkylating agent melphalan. Similarly, isatuximab enhanced the activity of different chemotherapies in an ALL tumor model. Its combination with vincristine, cytarabine or cyclophosphamide enhanced antitumor activity translating into a higher number of tumor-free survivors (TFS). These results document the unique MoA of isatuximab, primarily through ADCC and ADCP, and its robust antitumor activity as a single agent and in combination with standard of care treatments used in these hematological malignancies. ChemotherapyMM or T-ALL xenograft modelT/C IsatuximabT/C ChemotherapyT/C combinationPomalidomideMOLP-862%54%34%LenalidomideRPMI-822647%76%24%BortezomibNCI-H92928%26%0 %carfilzomibNCI-H92968%44%6%MelphalanLP-167%13%8%VincristineDND4141% no TFS0% no TFS0% 5/6 TFSAra-CDND4141% no TFS0% 3/6 TFS0% 6/6 TFScyclophosphamideDND4110% 4/7 TFS0% 4/7 TFS0% 7/7 TFS Citation Format: Chen Zhu, Celine Nicolazzi, Helgi Van de Velde, Dmitri Wiederschain, Sukhvinder Sidhu, Marielle Chiron. Isatuximab based combinations induce potent tumor growth inhibition in pre-clinical models of multiple myeloma and acute lymphocytic leukemia [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 5179.

Abstract 4816: Anetumab ravtansine has monotherapy efficacy in mesothelin positive patient-derived NSCLC tumor models and in a syngeneic tumor model in immunocompetent mice

Abstract Chemotherapy and immune checkpoint inhibitors (ICIs) are approved for treatment of non-small-cell lung cancer (NSCLC). However, there still remains a high medical need in NSCLC, eg. in patients non-responsive to ICIs or progressed after treatment with ICIs. Mesothelin (MSLN) is expressed in ~60% of lung adenocarcinomas. Here, we describe the mesothelin targeting antibody drug conjugate anetumab ravtansine (ARav) with the maytansinoid payload (DM4) as a novel treatment option for NSCLC. In the NSCLC cell line-derived xenograft model NCI-H322, ARav dosed at 2.5 mg/kg or 10 mg/kg, Q3Dx3, i.v., showed significant antitumor activity and was superior to cisplatin (dosed 3 mg/kg, Q3Dx12, ip). In addition, ARav monotherapy also showed antitumor activity in MSLN-positive NSCLC patient-derived xenograft (PDX) tumor models ST1243 and ST1684 at 15 mg/kg, Q2W. Maytansinoids and maytansinoid-based ADCs have been described to induce immunogenic cell death and immune response in vitro and in vivo, respectively. To explore the effects of ARav alone or in combination with anti PD-L1 Ab on tumor growth and the immune system, the MC38 C57BL/6 mouse colon cancer cell line was stably transfected with human mesothelin (MC38-hMSLN). MC38-hMSLN cells were transplanted s.c. in immunocompetent C57BL/6 mice to evaluate the anti-tumor activity of ARav. MC38-hMSLN had a high hMSLN expression level as shown by IHC. A dose of 10 mg/kg of ARav (Q3Dx3, i.v.) was highly efficacious with 11/12 animals showing complete regression of the tumor. Tumor free survivors (TFS) re-challenged 80 days after treatment all rejected MC38-hMSLN (11/11 animals), indicating the development of an immune memory response. The specificity of the immune response was further confirmed in an independently conducted experiment, where previously ARav treated MC38-hMSLN TFS mice were re-challenged with B16-F10 melanoma cells, which grew. Next, a lower dose of ARav (3 mg/kg, Q3Dx3, i.v.) was combined with an anti PD-L1 antibody in the MC38-hMSLN tumor bearing mice. The combination led to increased frequency of TFS compared to each monotherapy (12/12 TFS in the combination versus 7/12 for the anti PD-L1 and 2/12 for ARav). Further studies are currently ongoing to optimize the combination dosing and schedule as well as to characterize the immune cells involved in the response. In summary, the data supports the development of ARav in NSCLC and further exploration of ARav in combination with immune checkpoint inhibitors in MSLN-positive cancer indications. Anetumab ravtansine clinical activity is currently assessed at phase I studies in ovarian cancer in combination with pegylated liposomal doxorubicin (Phase 1b, NCT02751918), in multiple indications including NSCLC (Phase Ib, NCT03102320), and in combination with the anti PD-L1 antibody atezolizumab in NSCLC (Phase I /II, NCT03455556). Citation Format: Anette Sommer, Pascale Lejeune, Sabine Hoff, Annette O. Walter, Sandra Berndt, Lars Roese, Andreas Schlicker, Michael J. Wick, Cem Elbi, Dominik Mumberg, Christoph Schatz. Anetumab ravtansine has monotherapy efficacy in mesothelin positive patient-derived NSCLC tumor models and in a syngeneic tumor model in immunocompetent mice [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 4816.

Abstract 3718: ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules

Abstract Ovarian cancer (OC) is a gynecological malignancy with a high mortality rate of over 14,000 deaths annually in the US. Although ~ 80% of OC patients initially go into remission after 1st line treatment (surgery and chemotherapy), more than 60% relapse within 16-18 months. Low neoantigen burden and the immunologically “cold” nature of OC makes it a challenging malignancy. Therefore, novel methods such as immunotherapy and/or combined therapies are essential to improve clinical outcome of patients. This work evaluated the ID8-Luc murine ovarian carcinoma model to determine sensitivity to immunomodulatory agents. C57BL/6 albino mice implanted intraperitoneally with ID8-Luc cells were evaluated for growth and sensitivity to checkpoint inhibitors anti-PD-1, anti-PD-L1, anti-CTLA-4 and the chemotherapy agent paclitaxel. Disease progression was monitored weekly by in vivo bioluminescence imaging (BLI). Baseline immune profile of the model was determined by collecting ascites from untreated mice and analyzed with panels for myeloid and lymphoid cell populations by flow cytometry. ID8-Luc cells successfully established tumors with 7-8 days doubling time, as determined by BLI, and a median overall survival time of ~ 40-50 days. Formation of ascites resulted in weight gain and extended abdomen at advanced disease state. Necropsy showed solid tumor nodules within the peritoneal cavity including involvement of pancreas, liver, spleen and abdominal wall. Baseline immune profiling of ascites indicated presence of B cells (23%), granulocytic myeloid derived suppressor cells (G-MDSCs; 15%), TAMs (25%) and a low percentage of T cells (3-4%). This was suggestive of a possibly “cold” tumor model. Paclitaxel is a front-line chemotherapy option in OC. We find that the ID8-Luc model has 60% tumor free survivors (TFS) following treatment. To determine how this model responds to immune checkpoint inhibition we tested response to anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies. Treatment was initiated at either 7 (early) or 14 (late) days post tumor cell implant and we found that the overall response varied depending on the timing of treatment initiation. Early treatment with either anti-PD-1 or anti-PD-L1 resulted in complete regression of tumors (100% TFS) while the model was refractory to anti-CTLA-4 treatment. Late treatment initiation of anti-PD-1 or anti-PD-L1 resulted in an “all or nothing” response (40% complete response and 60% non-response). In addition, reducing the antibody dose from 10mg/kg to 5mg/kg did not make a material impact on the anti-tumor activity of these agents in the late treatment regimen. New models to further the preclinical testing of agents for OC is vital. This work characterizes the baseline immune profile of the murine ID8-Luc model along with response to checkpoint inhibitors to enable the rational design of combination strategies in the preclinical setting. Citation Format: Sumithra Urs, Sheri Barnes, Stacey Roys, Maryland R. Franklin. ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules [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 3718.

Abstract 2244: DeepIL-15 primed T cells synergize with PD-L1 blockade to overcome resistance to checkpoint immunotherapy

Abstract Introduction Interleukin-15 (IL-15) activates and expands both CD8+T cells and NK cells but not immunosuppressive Tregcells. While IL-15 is an attractive asset for cancer immunotherapy, its systemic administration is limited by toxicities. To limit IL-15 systemic exposure, we have developed DeepTM IL-15, a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc). Deep IL-15 is surface anchored to tumor reactive T cells prior to adoptive cell transfer (ACT). This novel therapeutic approach enables Deep IL-15 loading onto cells at concentrations unachievable with systemic IL15-Fc, causes autocrine T cell activation and expansion, yet limits systemic exposure and associated toxicities. The anti-tumor activity of T cell therapies has been limited by insufficient T cell expansion and by checkpoint immunosuppression. Here, we combined Deep IL-15 primed T cells with PD-L1 blockade to overcome these limitations. Methods Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinker. PMEL CD8+T cells (PMEL) were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice,activated, expanded, and Deep IL-15 was anchored to PMEL to generate Deep IL-15 Primed PMEL (Deep-15 PMEL). B16-F10 tumor-bearing mice were treated with cyclophosphamide (4 mg/mouse) one day prior to injection of 107Deep-15 PMEL (carrying ~ 58 ug of IL15-Fc)± anti-PD-L1 antibody (10 mg/kg q2w). We monitored mouse weight, tumor volumes, transferred and endogenous T cells, IL15-Fc systemic exposure, and cytokine release. Results Deep-15 PMEL showed significantly improved expansion and anti-tumor activity compared to PMEL, and provided long-term protection against tumor re-challenge. Importantly, the toxicity profile of Deep-15 PMEL was better than that of IL15-Fc co-injection with PMEL, yielding lower systemic exposure to IL15-Fc, less IFN-γinduction and no expansion of endogenous CD8+T and NK cells. Deep-15 PMEL resulted in 2/10 partial regressions and a statistically significant tumor growth inhibition compared to both vehicle and PD-L1 blockade alone. Co-administration of anti-PD-L1 further improved the anti-tumor activity of Deep-15 PMEL, resulting in 10/10 Complete Regressions (CR) and 6/10 Tumor Free Survivors. Conclusions By loading tumor-reactive T cells with Deep IL-15 before ACT, Torque's proprietary Deep Priming technology leverages the immune-stimulating power of IL-15 while avoiding immunotoxicity. Deep IL-15 Priming promotes engrafted T cell-specific activation and proliferation, and a favorable toxicity profile. In vivo transfer of Deep-15 PMEL combined with checkpoint inhibition was well tolerated and elicited remarkable anti-tumor activity in the PD-L1 refractory B16-F10 model, with 10/10 CRs. Torque is initiating a Phase I trial for TRQ15-01 (Deep IL-15 Primed multi-targeted human T cells) and planning a combination arm with checkpoint blockade in solid tumors. Citation Format: Elena Geretti, Xiaoyan Liang, Jesse Lyons, Philip Bardwell, Santina Caruso, Becker Hewes, Jonathan Fitzgerald, Karsten Sauer, Thomas Andresen. DeepIL-15 primed T cells synergize with PD-L1 blockade to overcome resistance to checkpoint immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2244.

Abstract 234: Pre-clinical characterization of 3A4-PL1601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) directed against KAAG1-expressing malignancies

Abstract Kidney-associated antigen 1 (KAAG1) is an 84 amino acid protein encoded by the reverse strand of a housekeeping gene called DCDC2 which was identified from a cDNA library derived from a histocompatibility leukocyte antigen-B7 renal carcinoma cell line as an antigenic peptide presented to cytotoxic T lymphocytes. More recently, KAAG1 has been identified by a sensitive subtractive cloning technology called STAR as a novel tumor-associated antigen expressed in a high percentage of ovarian tumors and triple-negative breast cancers (TNBCs), while it has restricted normal tissue expression. 3A4 is a humanized antibody raised against human KAAG1, which binds to KAAG1 expressed on the surface of cancer cells, rapidly internalizes and co-localizes with LAMP-1, a lysosomal marker. Altogether, KAAG1 represents an attractive target for an antibody-drug conjugate (ADC) approach based on its high and selective expression in various malignancies and its restricted expression in healthy tissues. 3A4-PL1601 is an ADC composed of the 3A4 antibody, site-specifically conjugated using GlycoConnectTM technology to PL1601, which contains HydraspaceTM, a valine-alanine cleavable linker and the PBD dimer cytotoxin SG3199. The purpose of this study was to characterize the in vitro and in vivoanti-tumor activity of 3A4-PL1601 in human cancer cell lines and xenografts models and to determine its safety and tolerability in the cynomolgus monkey. In vitro, the 3A4-PL1601 demonstrated potent cytotoxicity in a panel of human cancer cell lines of different origin and levels of KAAG1, while its potency was strongly reduced in KAAG1-negative cell lines. In vivo, 3A4-PL1601 showed potent and specific anti-tumor activity in the TNBC-derived MDA-MB-231 xenograft compared to the vehicle- and isotype control ADC-treated mice. A single dose of 3A4-PL1601 at 0.6 mg/kg resulted in 4/8 partial responders (PR) and 3/8 complete responders, two of which were tumor-free survivors at the end of the study on day 59, while none of the mice treated with the vehicle or the isotype-control ADC showed any activity. Moreover, 3A4-PL1601 showed potent and dose-dependent anti-tumor activity in the KAAG1-expressing, human renal cell carcinoma-derived SN12C xenograft when tested as single dose at 0.3, 0.6 or 1 mg/kg. At the highest dose tested, 3A4-PL1601 resulted in sustained tumor growth control and 2/8 PRs at the end of the study on day 60. 3A4-PL1601 was stable, well tolerated (MTD at least 0.8 mg/kg) and showed a favorable pharmacokinetic profile (t1/2 ~ 6 days) in the cynomolgus monkey (cross-reactive species). In conclusion, 3A4-PL1601 demonstrated potent and specific in vitro and in vivo anti-tumor activity in KAAG1-expressing cancer-derived models and it was stable and well tolerated in cynomolgus monkey, warranting further development of this ADC into the clinic. Citation Format: Francesca Zammarchi, Francois Bertelli, Karin Havenith, Ian Kirby, Simon Chivers, Patrick H. van Berkel. Pre-clinical characterization of 3A4-PL1601, a novel pyrrolobenzodiazepine (PBD) dimer-based antibody-drug conjugate (ADC) directed against KAAG1-expressing malignancies [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 234.