Ha Vo

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

Antibody-based therapies have revolutionized cancer treatment but have several limitations. These include downregulation of the target antigen, mutation of the target epitope, and, in the case of antibody-drug conjugates (ADC), resistance to the chemotherapy warhead. As TROP2-targeted therapy with ADCs yields responses in TROP2+ solid tumors, but the responses lack the durability observed with other immunotherapy-based approaches, we developed TROP2-targeting chimeric antigen receptor (CAR) T cells as an alternative. The TROP2-directed CAR T cells showed high potency against multiple solid tumor models. Moreover, TROP2-directed CAR T-cell therapy preserved high potency in models of ADC resistance and could be further engineered to prevent cell therapy resistance. This was achieved by leveraging fully human single-domain (VH-only) binder discovery to rationally engineer dual epitope binding-based (biparatopic) CARs. This work highlights the potency of CAR T-cell therapies and how rational engineering leveraging dual-VH targeting domains can overcome resistance pathways to current therapies. In future work, the CAR engineering approaches presented here can serve as a platform to be partnered with other strategies to address the suppressive tumor microenvironment.

Abstract Background N of 1 treatment paradigms represent the pinnacle of personalized medicine in which a patient’s tumors are profiled to guide treatment. Low-grade serous ovarian cancer (LGSC) is a distinct subtype of ovarian cancer, comprising ~10% of serous carcinomas and typically characterized by a younger age of onset. Molecularly, these tumors are often characterized by alterations within the Ras signaling pathway, including KRAS mutations. Clinically, LGSC is often resistant to standard cytotoxic chemotherapy, but may have sensitivity to hormonal therapy or MEK inhibitors. Here we report on a platform and proof of concept in one LGSC patient to evaluate personalized tumor-directed therapy regimens using patient-derived organoids (PDOs), BH3 profiling and viability evaluation in 3D microfluidic devices. Methods A patient with LGSC presented to the Dana Farber Cancer Institute and was treated with carboplatin and paclitaxel before a total abdominal hysterectomy with a bilateral salpingo-oophorectomy. Tissue was obtained under an IRB approved protocol and PDOs were established. Standard of care and non-standard of care treatments including doxorubicin, abemaciclib, letrozole, alpelisib, tamoxifen, trametinib, venetoclax, and navitoclax were evaluated by two orthogonal assays. First, they were tested for delta priming by BH3 profiling (Bhola et.al., Sci Signal. 2020 ) and second for cell viability using 3D microfluidic devices by TMRM/DRAQ7 dual-color fluorescent staining. Standard of care treatments carboplatin and paclitaxel were evaluated as individual treatments and in combination in 3D microfluidic devices. Results We successfully established a PDO model from the patient’s tumor sample in 14 days. BH3 profiling at 24 hours and viability in 3D microfluidic devices after 6 days in treatment showed that from the eight tested drugs, the model was sensitive to navitoclax and venetoclax. Average percent change in viability was -91.5% and -89.9%, respectively, and the drugs had a dynamic BH3 profiling index of 551.4 AUC (+/- 76.63) and 488.9 AUC (+/- 21.46) with the threshold of response being >175 for BH3 profiling. Trametinib showed a clear response in 3D, with an average percent change of -72.6% compared to the control but no significant response in BH3 profiling. Neither carboplatin and paclitaxel alone, nor in combination, elicited a significant change in viability. This observation was consistent with the patient’s history prior to surgery, where the tumor did not demonstrate significant clinical response to neoadjuvant carboplatin and paclitaxel therapy. Conclusions We describe a proof of concept of a N of 1 response assessment platform for LGSC using PDOs, BH3 profiling and live/dead fluorescent staining in microfluidic devices and demonstrate that BH3 profiling and 3D viability assessment assays show good congruity. Citation Format: Brittany Meisenheimer, Ha V. Vo, Kelley E. McQueeney, Aisha L. Saldanha, Carina Feeney, Courtney H. Qi, Swati Narayan, Jennifer D. Curtis, Marisa R. Nucci, Anthony Letai, Cloud P. Paweletz, Joyce F. Liu, Ursula A. Matulonis, Elena Ivanova. Individualizing treatment using patient derived organoids, BH3 profiling and microfluidics: A proof of concept in a patient with low-grade serous ovarian carcinoma [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 162.