Induce Tumor Cell Killing Research Articles

TRLS-06. PHASE 1 STUDY OF THE CD40 AGONISTIC MONOCLONAL ANTIBODY APX005M (SOTIGALIMAB) IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS WITH RECURRENT OR PROGRESSIVE CNS TUMORS AND NEWLY DIAGNOSED BRAIN STEM GLIOMA: A PEDIATRIC BRAIN TUMOR CONSORTIUM STUDY (PBTC-051; NCT03389802)

Abstract BACKGROUND CD40 is a co-stimulatory receptor present on antigen-presenting cells. CD40 agonists are hypothesized to reverse cancer-induced immune suppression and induce tumor cell killing. The PBTC completed a first-in-pediatrics phase 1 study (PBTC-051) of the CD40 agonistic monoclonal antibody APX005M (sotigalimab). METHODS Patients 1-21.99 years old with recurrent/progressive/refractory malignant non-brainstem central nervous system (CNS) tumors were eligible for stratum 1, and those with post-radiation pre-progression diffuse intrinsic pontine glioma (DIPG) for stratum 2. Patients received APX005M (sotigalimab) via IV on day 1 of each 21 day cycle. A 3 + 3 statistical design was used for dose escalation through 3 planned dose levels (DL). Patients could receive APX005M (sotigalimab) for 36 cycles or until disease progression, unacceptable toxicity, or death. RESULTS 31 eligible patients enrolled (20 on stratum 1, 11 on stratum 2) between 2018-2023; 29 were evaluable for dose finding. The most common diagnosis in stratum 1 was ependymoma (35%). DL3 (0.6 mg/kg) was the recommended phase 2 dose (RP2D) for stratum 1 and DL2 (0.3 mg/kg) for stratum 2. Five patients had dose limiting toxicities—two stratum 1 patients at DL3 and three stratum 2 patients at DL3. The most common grade 3+ adverse events at least possibly attributable to APX005M (sotigalimab) were lymphopenia, neutropenia, leukopenia, and increased ALT. Median number of cycles was 2 (1-36) for stratum 1 and 3 (1-19) for stratum 2. No patients had an objective response to APX005M. 6 month progression-free survival (PFS) for stratum 2 patients (calculated from start of treatment) was 30.7±12.8%; median PFS was 3.7 months. CONCLUSIONS APX005M was well-tolerated in pediatric patients with both recurrent/refractory/progressive CNS tumors and post-radiation pre-progression DIPG. A RP2D was established for both populations. Future studies of this agent in pediatric neuro-oncology patients should prioritize combination therapy.

Abstract 5277: The ROCK-2 inhibitor belumosudil exerts a direct antimyeloma effect and improves isatuximab-mediated cytotoxicity against multiple myeloma cells

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by clonal accumulation of malignant plasma cells in the bone marrow. Isatuximab (Sarclisa®) is a monoclonal antibody used for the treatment of MM. Isatuximab binds to CD38 (highly expressed on MM cells) and induces tumor cell killing via several mechanisms including antibody dependent cellular cytotoxicity (ADCC) and direct apoptosis. Belumosudil (RezurockTM) is a ROCK2 inhibitor that is approved for the treatment of chronic graft versus host disease (cGVHD). Recent reports suggest that inhibition of ROCK2 induces apoptosis and prevents growth of MM cells. Here, we investigated the potential antimyeloma effect of belumosudil alone or in combination with isatuximab with different readouts of viability, apoptosis and cytotoxicity. The potential cytotoxic effect of belumosudil (as single agent or in combination with isatuximab) against MM cells was assessed in vitro by flow cytometry apoptosis assay, CellTiter-Glo® luminescent cell viability assay or long-term cytotoxic assay (Incucyte). The effect of belumosudil on healthy donor-derived NK cells viability was also assessed by flow cytometry. Belumosudil reduced the number and viability of several MM cell lines without affecting healthy donor NK cell number and viability. Moreover, belumosudil induced apoptosis of MOLP-8 MM cells in a dose-dependent manner and further increased MOLP-8 cell apoptosis when combined with isatuximab after 2, 3 and 4 days of in vitro treatment. Belumosudil induced killing of MM cells in a dose-dependent manner over time (up to 4 days), independently of the presence of NK cells, as measured by Incucyte-based cytotoxicity assay. In addition, the combination of belumosudil (3.3 µM) with isatuximab (10 µg/ml) in presence of NK cells enhanced the killing of different MM cell lines over time compared to single agents.Finally, the expression of CD38 on MM cells has been documented to be decreased after treatment with anti-CD38 antibodies. Interestingly, analysis of CD38 expression on MM cells indicated that belumosudil induces an increase of CD38 on the surface of MOLP-8 MM cells and prevents isatuximab-induced decrease of CD38 expression. These data suggest that belumosudil has a direct antimyeloma effect and increases isatuximab-mediated cytotoxicity against MM cells, without affecting the viability of NK cells. Taken together, this study provides evidence of the therapeutic potential of belumosudil in combination with isatuximab in the setting of MM. Citation Format: Marco Meloni, Pauline Perrin, Claire Nouguier, Stephane Poirier, Monsif Bouaboula, Kamlesh Bisht, Helgi Van de Velde, Angela Virone-Oddos, Marielle Chiron. The ROCK-2 inhibitor belumosudil exerts a direct antimyeloma effect and improves isatuximab-mediated cytotoxicity against multiple myeloma cells [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 5277.

Abstract 6724: Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model

Abstract In the field of cancer immunotherapy, bispecific antibodies have emerged as a promising approach for redirecting T-cells to target and combat tumors. However, their efficacy is limited to only a subset of patients. A major challenge that hinders the development of bispecific antibody targets is the lack of preclinical models that preserve patient-specific tumor antigens and recapitulate the complex interaction between the human immune system and tumor cells. HUB Organoids technology presents a valuable platform for novel immunotherapies testing and validation as they are directly derived from patient tumor tissues, accurately reflecting patient-specific tumor antigens. BIOMUNEX Pharmaceuticals has developed the BiXAb® technology platform, a computational modeling approach that rapidly generates bispecific antibodies from various monospecific monoclonal antibodies, eliminating the need for extensive engineering. This platform includes a bivalent T cell engager specifically targeting MAIT cells and human epithelial growth factor receptor (HER2), a tumor antigen highly expressed in some colorectal cancer (CRC) patients. This innovative approach forms an efficient immunological synapse, allowing exclusive redirection of MAIT cells to directly eliminate cancer cells. In a collaborative research initiative between HUB Organoids® and BIOMUNEX Pharmaceuticals, the mechanisms by which BiXAb® induce tumor cell killing were confirmed using CRC patient-derived organoids (HUB Organoids® or PDOs) co-cultured with allogenic MAIT cells in an in vitro model. The study results depicted BiXAb® induced MAIT-cell activation in a dose-dependent manner, as detected by FACS and ELISA. Furthermore, BiXAb®-mediated MAIT-cell-specific killing of CRC PDOs was observed in a HER2-dependent manner, determined using an image-based viability assay. In conclusion, the co-culture platform of CRC PDOs-MAIT cells described here effectively captured the activation and targeted killing of MAIT cells mediated by BiXAb®, underscoring the added value of this PDO-based co-culture platform in developing novel immunotherapies. Citation Format: Yasmine Abouleila, Julie Prigent, Mayke Doorn, Claire Germain, Sylvia Boj, Simon Plyte, Carla Verissimo. Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model [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 6724.

Abstract 4224: The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment

Abstract We developed an in vitro protocol to generate tumor-specific cytotoxic lymphocytes (CTL) by priming T cells from healthy donors on HER2+ breast cancer cell line SKBR-3. Subsequently, these primed CTLs were tested in a 3D immune cell killing assays in a life cell imaging device. An image analysis workflow was applied to understand the T cell movement in spatial relation to the tumor and the kinetic of tumor cell killing. The activity of the CTLs was compared with non-activated CD8+T cells, staurosporin and CD8+ T cells, unspecifically activated by αCD2/αCD3/αCD28 beads. The tumor cells were expressing mKate whereas the T cells were label-free. By analyzing a combination of different masks, we could computationally measure tumor spheroid characteristics and dynamic attributes such as changes in shape, percent of cells expressing red fluorescentce, and relative average distance of T cells from the spheroid in each timelapse assay. This process was applied at each frame to determine the average T cell distance from its corresponding spheroid center and subsequently calculate the average T cell flux over the course of each assay by linking data between image frames. Additional measurements allowed for the quantification of tumor debris accumulation over time both relative to the primary tumor mass and in absolute terms. Our analyses revealed that bead-activated T cells approached the tumor spheroid with the highest speed and infiltrated the spheroid homogeneously. In contrast the CTLs approached the spheroid slower and started tumor cell killing from the rim of the spheroid towards the center. The reduction of spheroid area over time displayed a very similar kinetic when co-cultured with CTL or bead-activated T cells. At the end of the experiment the CTLs induced a more pronounced shrinkage of spheroid size. Interestingly the size of the spheroid and the red fluorescence intensity were not necessarily aligned. The positive control Staurosporin induced a fast reduction of the mKate signal, whereas the spheroid area did not change over time. In summary, the bead-activated T cell approached the spheroid fast, infiltrated the complete area and induced tumor cell killing homogenously across the tumor area. The CTL approached the spheroids slower but started killing earlier from the outside towards the center of the spheroid. The observed differences in infiltration and killing characteristics of the investigated T cell types lead to a better understanding of T cell biology in the context of the tumor biology, which can be translated into applications during dug development. Taken together, with the applied image analysis algorithm we were able to deconvolute the biological processes in the assay in much more detail. The additional read-outs increase the translational value of the assay tremendously in the context of drug development as well as precision medicine. Citation Format: Ryan Kerwin, Ina Rohleff, Martin Blanchard, Kanstantsin Lashuk, Bryan Walker, Julia B. Schueler. The application of sophisticated image analysis to a T cell killing assay in 3D deciphers the dynamics of the different components of the tumor microenvironment [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 4224.

Abstract 2375: 23ME-01473, a novel anti-ULBP6/2/5 monoclonal antibody, reinvigorates anti-tumor NK cell function through NKG2D and FcγRIIIa activation

Abstract Using genome-wide association studies (GWAS) of the 23andMe genetic and healthy survey database across all phenotypes as part of our unique target discovery platform, we identified immuno-oncology (I/O)-related genetic variants as those with opposing directionality of effects on cancer and immunological phenotypes, which comprise the I/O signature. This I/O signature identified ULBP6 as a potential cancer therapeutic target. ULBP6 is a stress-induced cell surface ligand found on cancer cells, that binds to the activating receptor, NKG2D, on NK and T cells to induce tumor cell killing. However, as a mechanism of immune escape, tumors shed ULBP6 to produce a soluble form that attenuates NKG2D activation and is elevated in cancers. Given that the 2 allelic variants of ULBP6 have the highest binding affinity to NKG2D of all human NKG2D ligands, ULBP6 may be the most immunosuppressive soluble NKG2D ligand and a critical regulator of anti-tumor response. Accordingly, soluble ULBP6 exhibited significant suppression of IFNγ release and detectable cell surface NKG2D expression on healthy donor PBMCs using in vitro cell-based assays. Moreover, the overexpression of human ULBP6 in a genetically modified MC38 murine model resulted in decreased infiltration of NK, NKT, and CD8 T cells in vivo. As such, we developed a novel humanized monoclonal antibody, 23ME-01473, that binds with high affinity to ULBP6, and due to the high sequence homology, to ULBP2 and ULBP5, to block their interaction with NKG2D. By preventing the binding of soluble ULBP6 to NKG2D on PBMCs in vitro, 23ME-01473 restored NKG2D activation as measured by increased detectable cell surface NKG2D expression, IFNγ and granzyme B release, and tumor cell killing by PBMCs. To further leverage the binding of 23ME-01473 to ULBP6/2/5, 23ME-01473 was designed to have an afucosylated Fc domain that enhances its binding affinity to the activating Fc receptor, FcγRIIIa, on NK cells to induce ADCC against tumor cells. The combination of NKG2D and FcγRIIIa activation resulted in synergistic PBMC-mediated IFNγ secretion and enhanced tumor cell killing, compared to activation of either NKG2D or FcγRIIIa. Taken together, these results reveal ULBP6 as a novel cancer therapeutic target, and given the dual activation of NKG2D and FcγRIIIa, suggest the potential of 23ME-01473 to activate NK cells and elicit anti-tumor immunity. Citation Format: Joel Benjamin, Abigail Jarret, Shashank Bharill, Shruti Yadav, Dina Ayupova, Clifford Hom, Zahra Bahrami Dizicheh, I-Ling Chen, Anh Diep, Shi Shi, Caroline Bonnans, Danielle Kellar, Germaine Fuh, Maike Schmidt, Kimberline Gerrick, Patrick Koenig, Mauro Poggio. 23ME-01473, a novel anti-ULBP6/2/5 monoclonal antibody, reinvigorates anti-tumor NK cell function through NKG2D and FcγRIIIa activation [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 2375.

Abstract 812: Treatment of metastatic cancers using immunotherapeutic cowpea mosaic virus nanoparticles

Abstract This study investigates the use of an immunotherapeutic plant virus, namely the cowpea mosaic virus (CPMV), that when used prophylactically and therapeutically can protect against metastatic cancer in multiple tumor mouse models. Metastatic cancer continues to be the main cause of death of most cancer patients, and new treatment modalities must be engineered to protect patients from metastatic disease. For instance, peritoneal metastases (PMs) are oftentimes fatal, and patients survive for a median 6 months following diagnosis. PMs are common for colon, ovarian, gastric, appendiceal, and pancreatic cancers. Previously, it was demonstrated that CPMV is a potent in situ vaccine causing tumor shrinkage with abscopal effect and long-term protection observed in tumor mouse models and canine cancer patients. CPMV functions as a toll like receptor (TLR) agonist and acts on innate immune cells to induce tumor cell killing and antigen processing to eventually prime adaptative and tumor-specific immunity. However, metastatic cancers such as PMs are not easily and directly injectable and therefore, we have expanded the ability of CPMV to protect against PMs by engineering targeted CPMV nanoparticles. Data indicates that CPMV can improve survival and slow tumor growth in a murine colon PM model compared to controls and that long-term survivors are further protected from re-challenge indicating tumor specificity. Analysis from current and past studies indicate that CPMV injection into the intraperitoneal space leads to distinct upregulation of immunostimulatory cytokines and recruitment of innate immune cell populations. PMs lead to extreme declines in quality of life of patients, and current treatments can be ineffective in slowing down or reducing tumor development and death - in tackling this problem, the data clearly points to the strong potential for CPMV-mediated therapeutics in the treatment and prophylaxis of PMs. Dr. Steinmetz is a co-founder of and has a financial interest in Mosaic ImmunoEngineering Inc. The other authors have no conflict of interest to declare. Acknowledgments: This work is supported in part through NIH grants R01 CA274640, R01 CA224605, R01 CA253615, CDMRP W81XWH2010742, and the Shaughnessy Family Fund for Nano-ImmunoEngineering (nanoIE at UC San Diego). Citation Format: Young Hun Chung, Britney A. Volckaert, Nicole F. Steinmetz. Treatment of metastatic cancers using immunotherapeutic cowpea mosaic virus nanoparticles [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 812.

Abstract 4131: Long-term anti-tumor immunity induced by of HPN328, a DLL3-targeting trispecific, half-life extended T cell engager, in a preclinical immunocompetent mouse model

Abstract Tri-specific T Cell-Activating Constructs (TriTACs) are T-cell engagers that have been developed to redirect T cells to kill tumor cells. HPN328 is a Delta-like ligand 3 (DLL3)-targeting TriTAC currently being evaluated in a Phase 1/2 clinical trial enrolling patients with advanced cancers associated with DLL3 expression, including small cell lung cancer (SCLC) and other neuroendocrine malignancies (NCT04471727). HPN328 consists of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. We have previously reported that HPN328 induces potent dose-dependent killing of DLL3 expressing SCLC cell lines together with dose-dependent T cell activation and cytokine release in T cell co-cultures in vitro. Based on these data and the proposed mechanism of TriTAC induced tumor cell killing we hypothesized that HPN328 could induce epitope spreading and long-term immunogenic effects. To test this hypothesis, we generated an immunocompetent mouse model in which six amino acid residues corresponding to the portion of human CD3ε which TriTAC molecules bind were added to the N terminus of the mouse CD3ε subunit. Homozygous knock-in mice (hCD3ε) were implanted subcutaneously with MC38 murine colon cancer cell line expressing human DLL3 and treated with HPN328 following tumor establishment. HPN328 treatment led to eradication of established tumors in mice. In addition, tumor infiltrating lymphocyte (TIL) analysis was performed on tumors harvested on day 4 post HPN328 treatment. Activation markers CD69 and CD25 were induced on both CD4+ and CD8+ TILs in this model. To evaluate if HPN328 can induce long-term immunity, mice whose tumors were cured by HPN328 were rechallenged on the opposite flank. As expected, tumors grew in treatment naïve mice, but no tumors were detected in mice previously treated with HPN328, demonstrating sustained and durable anti-tumor immunity induced by HPN328 treatment. Anti-tumor immunity is an indicator of T cell memory against tumor antigens. Concomitant with anti-tumor immune response, we detected increases in memory T cells in the spleens of mice previously treated with HPN328. Together these results indicate that HPN328 can induce epitope spreading and prolonged anti-tumor immunity, suggest a novel mechanism for its activity and efficacy in vivo. Citation Format: Mary Ellen Molloy, Laura B. Valenzuela, Chi-Heng Wu, Holger Wesche, Banmeet S. Anand. Long-term anti-tumor immunity induced by of HPN328, a DLL3-targeting trispecific, half-life extended T cell engager, in a preclinical immunocompetent mouse model. [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 4131.

Abstract 1803: GNP101, anti-HER2-IFN-β mutein immunocytokine, for the treatment of Trastuzumab deruxtecan-resistant HER2-positive cancers

Abstract Background: Trastuzumab deruxtecan (T-DXd) has been approved in various types cancer and recently expanded its indication to cancers with various HER2 expressions, such as HER2-low breast cancer. Since the effect of T-DXd is based on toxin payload, topoisomerase I inhibitor, the emergence of toxin-resistant cancer cells is unavoidable. Clinical cases of Primary, or secondary resistance to T-DXd have been reported. Therefore, it is necessary to develop novel drugs that can effectively exhibit antitumor effects against T-DXd-resistant carcinoma. Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. It is one of the few cytokines that induce tumor cell killing directly. Importantly, it could be an alternative to conventional drug-resistant cancer, because it regulates tumor growth in a way different from cytotoxic agents. Here, GNP101, an IFN-β mutein-based immunocytokine, exhibits antitumor properties against HER2-positive cancers, including T-DXd-resistant cancer. Method: A panel of human HER2-positive cancer cell lines derived from breast, stomach and lung were treated with GNP101 or HER2 therapeutics (trastuzumab, T-DM1, T-DXd) to compare direct antitumor effects. To generate a T-DXd-resistant cells, HCC1954, NCI-N87 and Calu-3 were exposed to the IC50, IC80 or IC90 of T-DXd by cyclic treatment. After 4 months, HCC1954-R cells were established and IFN-β signaling was assessed by STAT1-phosphorylation. The direct antitumor effect of GNP101 in vivo was tested in HER2-positive cancer xenograft models. Result: GNP101 showed superior potency compared to existing HER2 therapies (trastuzumab, T-DM1, and T-DXd) in all HER2-positive cancers regardless of tissue origin. To investigate the antitumor effect of GNP101 against T-DXd resistant cancer, we first generated T-DXd-resistant cell line (HCC1954-R). These HCC1954-R cells were strongly resistant to T-DXd, and showed no antitumor effect even at 100 nM of T-DXd. However, they were sensitive to GNP101, suggesting that GNP101 maintained its antitumor effect in T-DXd resistant cells. In addition, there was no difference in IFN-β-induced STAT1 phosphorylation between the parent cell and HCC1954-R. Taken together, these results indicate that HCC1954-R cells maintain IFN-β signaling and are selectively resistant to T-DXd. The activities of GNP101, T-DM1 and T-DXd on HER2-positive cancer cells were compared in vivo. GNP101 inhibited the growth of tumor by 90%, with no difference from that of T-DXd or T-DM1. Conclusion: We have evaluated the antitumor properties of GNP101, a trastuzumab-IFN-β mutein in HER2-expressing cancer. Given that GNP101 retains its antitumor properties in T-DXd-resistant cancer cells, the study suggests that anti-HER2-IFN-β mutein is a promising candidate for the treatment of HER2-positive and T-DXd resistant cancers. Citation Format: Chan Gyu Lee, Minkyung Park, Hyun Kyung Lee, Na Young Kim, Jun Young Choi, Sungyoul Hong, Young Kee Shin, Hae Min Jeong. GNP101, anti-HER2-IFN-β mutein immunocytokine, for the treatment of Trastuzumab deruxtecan-resistant HER2-positive cancers [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 1803.

Identification of a subset of tumor-reactive CD8 TILs expressing NKG2A in HNSCC and CRC

Abstract Even though blocking the interaction of NKG2A with its ligand HLA-E is a promising strategy to restore the function of cytotoxic T cells and induce tumor cell killing, several aspects of the biology of NKG2A+ CD8 T cells remain to be understood to fully benefit from this therapeutic approach. Here, we characterize the expression of NKG2A on tumor-infiltrating CD8 T cells (CD8 TILs) in patients with head and neck squamous cell carcinoma (HNSCC) and microsatellite-stable colorectal cancer (CRC). Our results show that NKG2A is primarily expressed by a population of tumor-reactive CD8 TILs identified by coexpression of CD39 and CD103 (DP CD8). NKG2A expression was stable and maintained during in vitro expansion. Interestingly, while TCR stimulation in presence of IL-15 and TGF-β are thought to be responsible for NKG2A upregulation, our in vitro results suggest that other factors might be necessary to induce its expression on naive CD8 T cells. Ex vivo analysis of NKG2A+ DP CD8 TILs by scRNAseq and flow cytometry revealed that those cells were activated, more differentiated, and displayed a cytolytic profile suggesting a role in tumor recognition and killing. Finally, we found that, even though there was a high degree of clonal overlap between NKG2A+ and NKG2A− DP CD8, some CDR3 TCR sequences were enriched in one versus the other subset. Consequently, HPV-reactive cells were preferentially enriched in NKG2A− DP CD8 TILs. Altogether, our results provide evidence that NKG2A is expressed by tumor-reactive CD8 TILs in HNSCC and CRC. However, NKG2A-inducing factor(s) remain to be identified and might provide new potential drug targets to bolster anti-tumor immune responses in cancer patients. Supported by the Providence Portland Medical Foundation.

Peptide hydrogels loaded with irradiated tumor cell secretions enhance cancer immunotherapy

In advanced stage patients, traditional cancer chemotherapies often demonstrate limited tumor growth inhibition and fail to elicit sufficient anti-tumor immunity required for long-lasting responses. Herein, we developed a therapeutic peptide hydrogel manufactured using a PEG cross-linked melittin-peptide scaffold that not only itself induces tumor cell killing, but also encapsulates the processed cell secretions of autologous, irradiated tumor cells. MLT-RADA-Polyethylene glycol (MRP) loaded with the concentrated ultrafiltration (UF) retentate from irradiated tumor cell media, denoted as UF@MRP effectively reshapes the tumor microenvironment (TME) by stimulating dendritic cell maturation and reprogramming macrophages toward a tumoricidal M1 phenotype. In multiple murine tumor models, in which immune checkpoint blockade (ICB) alone did not control advanced disease, intratumoral administration of UF@MRP enhanced ICB efficacy, leading to prolonged survival. The combination of UF@MRP and ICB appears to be an effective approach to rapidly kill tumor cells, diminish immunosuppression in the TME, and activate host adaptive immune responses, all of which work in concert in an immunotherapy regime to control established and aggressive tumors.

Abstract 2712: Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers

Abstract The success of early cancer immunotherapies has led to the development of novel therapeutic approaches including multi-specific antibodies engaging innate and adaptive effector cells. To evaluate the antitumor potency of these multi-specific cell engagers, we have established a protocol to select human PBMCs from donors of a commercial frozen source by their potency to produce inflammatory cytokines and the capacity to engraft into NOD-Scid IL2Rgammanull (NSG)mice. The selected human PBMCs were used to reconstitute NSG mice, or as donors of effector cells that upon in vitro expansion were adoptively transferred to NSG mice for humanization. We have employed these humanization strategies to evaluate T cell engagers, which are typically multi-specific antibodies directed against the T cell and one or multiple tumor-associated antigen (TAA), whose therapeutic strategy is to 1) engage T cells, 2) activate the T cells and 3) engage tumor cells, and thus induce tumor cell killing. We will present representative data from several humanization approaches using liquid and solid orthotopic tumor models to assess the effectiveness of multi-specific T cell engagers in mediating antitumor responses. Thus, pre-clinical evaluations rely on the development of optimized tumor bearing humanized models that mirror key properties of a human setting, to assess the therapeutic potential of anti-tumor multi-specific biologics. Citation Format: Liqing Cheng, Jane Chen, Qunyan Yu, Natalia Malkova, Virna F. Cortez-Retamozo, Fangxian Sun, Sukhvinder Sidhu. Donor selection strategies to optimize humanized mouse models for the evaluation of T cell engagers [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 2712.

Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects.Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses.Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells.Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma

AbstractIn relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-programmed death-1 inhibitor pembrolizumab have demonstrated efficacy as monotherapy and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody, designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have demonstrated signs of efficacy as monotherapy for patients with R/R HL and the combination of AFM13 with pembrolizumab represents a rational new treatment modality. Here, we describe a phase 1b, dose-escalation study to assess the safety and preliminary efficacy of AFM13 in combination with pembrolizumab in patients with R/R HL. The primary objective was estimating the maximum tolerated dose; the secondary objectives were to assess safety, tolerability, antitumor efficacy, pharmacokinetics, and pharmacodynamics. In this heavily pretreated patient population, treatment with the combination of AFM13 and pembrolizumab was generally well tolerated, with similar safety profiles compared to the known profiles of each agent alone. The combination of AFM13 with pembrolizumab demonstrated an objective response rate of 88% at the highest treatment dose, with an 83% overall response rate for the overall population. Pharmacokinetic assessment of AFM13 in the combination setting revealed a half-life of up to 20.6 hours. This proof-of-concept study holds promise as a novel immunotherapy combination worthy of further investigation. This phase 1b study was registered at www.clinicaltrials.gov as NCT02665650.

Isatuximab Acts Through Fc-Dependent, Independent, and Direct Pathways to Kill Multiple Myeloma Cells.

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-β) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.

Abstract 2244: PARP inhibition in combination with pembrolizumab enhances cytotoxicity in ovarian cancer patient-derived 3D spheroids

Abstract Poly(ADP-ribose) polymerase (PARP) inhibitors have shown impressive clinical activities for ovarian cancer patients with BRCA mutations and have altered the paradigm for ovarian cancer treatment. Despite the utility that PARP inhibitors provide, intrinsic and acquired resistance often limit their effectiveness as a monotherapy, and therefore combination treatment is an attractive alternative for more durable responses. Checkpoint inhibitors have shown impressive efficacy in numerous solid tumor types, but have failed to show meaningful response in ovarian cancer. The role PARP inhibitors play as immune modulators to enhance checkpoint blockade efficacy has just recently emerged indicating that their combination with checkpoint inhibitors may be beneficial over either single agent. A recent phase I/II clinical trial by researchers at the Dana-Farber Cancer Institute demonstrated niraparib in combination with pembrolizumab produced complete or partial responses in 18% of patients with recurrent platinum-resistant ovarian cancer compared to less than a 5% response rate with PARP inhibitors alone (JAMA Oncol. 2019;5(8):1141-1149). Further understanding of the immune modulatory capacity of PARP inhibitors alone and in combination with checkpoint blockade will aid in the prediction of patient response or resistance to therapy. To address this lack in knowledge, we evaluated therapeutic responses to PARP inhibitors, checkpoint inhibitors, and combinations using ovarian cancer patient-derived 3D tumor spheroids. Primary tumor samples were evaluated for PD-L1 and EpCAM expression, and tumor infiltrating lymphocyte (TILs) populations were assessed for T-cell activation and CD8+:Treg ratios. Immune-mediated antitumor activity was investigated by monitoring changes in PD-L1 and MHC class-I expression in the tumor cell populations following PARP inhibition, pembrolizumab treatment, and in combination. The effects of pembrolizumab alone and in combination for enhanced T-cell mediated tumor cell killing were also tested. PD-L1 expression was associated with response to pembrolizumab, and pembrolizumab induced tumor cell killing was T-cell dependent. Despite detection of cytotoxic activity by olaparib and pembrolizumab as single agents, increased tumor cell killing was detected when olaparib and pembrolizumab were used in combination. In summary, we detected therapy related modulation of tumor cells as well as the immune composition which results in decreased tumor spheroid viability in the presence of PARP inhibition in combination with checkpoint blockade. These results highlight the potential benefit of PARP/checkpoint inhibitor combination therapy and of patient-derived 3D spheroids as a platform to help identify subsets of cancer patients who would likely respond to immunotherapies as single agents or in combination with other approved agents. Citation Format: Kathryn M. Appleton, Ashley Elrod, Stephen Shuford, Teresa M. DesRochers. PARP inhibition in combination with pembrolizumab enhances cytotoxicity in ovarian cancer patient-derived 3D spheroids [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 2244.

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.

Correction: LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

T Cell-Activating Bispecific Antibodies in Cancer Therapy.

Effector lymphocytes are multifunctional cells of the immune system that promote cytolysis of pathogen-infected cells and nascent tumors. Tumors must learn to evade effectors and employ a wide variety of mechanisms to do so. Bispecific Abs (BsAbs) are an emerging cancer immunotherapy approach seeking to re-engage either T effectors or NK cells with malignant cells. Possessing specificity for effector cells on one end and a tumor Ag on the other, these molecules work by attracting effectors to the target cell to build an immunologic synapse and induce tumor cell killing. The BsAb blinatumomab, for example, has specificity for the T cell-activating cell surface protein CD3 and the B cell Ag CD19. The only BsAb with regulatory approval currently, blinatumomab is used in the treatment of relapsed or refractory B cell acute lymphoblastic leukemia. Many additional BsAbs are in preclinical development, however, targeting many different tumor types. The variety of potential effector cells and cancer Ags, along with potential combination therapies, make BsAbs an active area of drug development. In this review, we discuss cancer recognition by the immune system and structural and mechanistic aspects of BsAbs. We summarize key steps in preclinical development and subsequent translation to medical practice. Future directions for BsAbs include combinations with a wide variety of both immunologic and nonimmunologic therapies. Defining their optimum clinical use is at early stages.

Abstract 2966: Isatuximab-induced multiple myeloma cell killing through effector functions is dependent on CD38 expression and complement inhibitors

Abstract Isatuximab (Isa) is an IgG1 monoclonal antibody (Ab) that specifically recognizes human CD38. Once Isa engages multiple myeloma (MM) cells expressing a high level of CD38, it can induce tumor cell killing via Fc-dependent mechanisms including Ab-dependent cell-mediated cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). To better understand the mechanism of Isa-mediated cytotoxicity, we studied CD38 levels in 16 established MM lines and measured Isa-mediated ADCC, ADCP, and CDC in tumor cell killing. The cytotoxic functions of Isa were dependent on CD38 receptor density (RD) in most cell lines. Isa-mediated ADCC was observed in a subset (7/16) of MM cell lines that displayed CD38 RD &amp;gt;100,000 molecules/cell. Similarly, the same subset of MM cells, with 1 exception, were sensitive to Isa-mediated ADCP, indicating a similar CD38 RD is also required for this killing effect. A much higher CD38 RD (&amp;gt;250,000 molecules/cell) alone was insufficient for Isa-mediated CDC. Cell lines MOLP-8 and MOLP-2 have CD38 RD &amp;gt;250,000 molecules/cell, but are resistant to Isa-mediated CDC. Overexpression of CD38 in cell lines with low endogenous CD38 expression did not always sensitize the cells to Isa-mediated CDC. These results suggest that additional mechanisms are involved in the regulation of such cytotoxic effects. We further investigated the expression of complement-cascade inhibitors CD46, CD55, and CD59. High-level expression of at least one of these molecules was associated with resistance to Isa-mediated CDC, even when cells were CD38 high (hi). By comparing 4 selected MM cell lines, we found that a minimal RD level of 50,000 molecules/cell for CD46, CD55 or CD59 appears to be an important threshold to suppress Isa-mediated CDC. When CD59 function was neutralized by an anti-CD59 antibody, we were able to re-sensitize the cells to Isa-mediated CDC in CD38hi MOLP-8 cells. Neutralizing CD59 function alone did not rescue Isa-mediated CDC in CD38-low expressing NCI-H929 cells. However, overexpressing CD38 and inhibiting CD59 rendered NCI-H929 cells sensitive to CDC. Taken together, the high-level of CD38 expression and low-level of CD59 (and perhaps other inhibitors) expression are important for Isa-mediated CDC in killing of target tumor cells. In conclusion, the main immune effector mechanisms involved in Isa-mediated killing of MM cells include ADCC and ADCP. These effects are dependent on high levels of CD38 RD in MM cell lines in vitro. Further confirmation is under way using samples from MM patients. Citation Format: Zhili Song, Guang Yang, Anlai Wang, Rita Greco, Joachim Theilhaber, Elvis Shehu, Daniel Ajona, Bruno Paiva, Chen Zhu, Dmitri Wiederschain, Marielle F. Chiron Blondel, Francisco Adrian. Isatuximab-induced multiple myeloma cell killing through effector functions is dependent on CD38 expression and complement inhibitors [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 2966.

TRAIL acts synergistically with iron oxide nanocluster-mediated magneto- and photothermia.

Targeting TRAIL (Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand) receptors for cancer therapy remains challenging due to tumor cell resistance and poor preparations of TRAIL or its derivatives. Herein, to optimize its therapeutic use, TRAIL was grafted onto iron oxide nanoclusters (NCs) with the aim of increasing its pro-apoptotic potential through nanoparticle-mediated magnetic hyperthermia (MHT) or photothermia (PT).Methods: The nanovector, NC@TRAIL, was characterized in terms of size, grafting efficiency, and potential for MHT and PT. The therapeutic function was assessed on a TRAIL-resistant breast cancer cell line, MDA-MB-231, wild type (WT) or TRAIL-receptor-deficient (DKO), by combining complementary methylene blue assay and flow cytometry detection of apoptosis and necrosis.Results: Combined with MHT or PT under conditions of “moderate hyperthermia” at low concentrations, NC@TRAIL acts synergistically with the TRAIL receptor to increase the cell death rate beyond what can be explained by the mere global elevation of temperature. In contrast, all results are consistent with the idea that there are hotspots, close to the nanovector and, therefore, to the membrane receptor, which cause disruption of the cell membrane. Furthermore, nanovectors targeting other membrane receptors, unrelated to the TNF superfamily, were also found to cause tumor cell damage upon PT. Indeed, functionalization of NCs by transferrin (NC@Tf) or human serum albumin (NC@HSA) induces tumor cell killing when combined with PT, albeit less efficiently than NC@TRAIL.Conclusions: Given that magnetic nanoparticles can easily be functionalized with molecules or proteins recognizing membrane receptors, these results should pave the way to original remote-controlled antitumoral targeted thermal therapies.