Tumor Clearance Research Articles

‘Thou shalt not throw away a living thing’: A single-centre 10-year experience using the spare parts principle for complex sarcoma reconstruction

For aggressive limb sarcomas beyond reconstructive reach, an amputation may be the only alternative to achieve a complete tumour resection. However, very proximal amputations result in greater loss of function and quality-of-life impact. The spare parts principle advocates utilising tissues distal to the amputation site, for reconstructing complex defects and preserving the function. We aim to present our 10-year experience utilising this principle in complex sarcoma surgery. A retrospective review of our prospective sarcoma database was conducted for sarcoma patients treated with an amputation between 2012 and 2022. Cases in which distal segments were used for the reconstruction were identified. Demographic data, tumour characteristics, and surgical and non-surgical treatment, along with oncological outcomes and complications, were recorded and analysed. Fourteen patients were eligible for inclusion. The median age was 54 years at presentation (8-80 years) with 43% being females. Nine had a primary sarcoma resection, two were treated for recurrent tumours, two presented intractable osteomyelitis following sarcoma treatment and one had an amputation as a palliative procedure. The latter was the only oncological case in which tumour clearance was not achieved. Three patients developed metastasis and subsequently died during follow-up. Careful balancing of oncological goals and preservation of function is required for proximal limb-threatening sarcomas. When an amputation is required, tissues distal to the cancer site provide a safe reconstructive alternative, optimising patient recovery and preserving function. Our experience is limited by the small number of cases presenting with these rare and aggressive tumours.

Abstract CT234: A phase I/II trial investigating safety and efficacy of autologous TAC01-HER2 in relapsed or refractory solid tumors

Abstract Background: Despite recent therapeutic developments for patients with advanced, metastatic, unresectable HER2+ solid tumors, significant unmet medical needs still exist, especially in tumors other than breast and gastric. The T cell antigen coupler (TAC) technology is a novel approach to modifying T cells, allowing them to recognize and treat HER2+ solid tumors. The TAC receptor redirects T cells to tumor cells, and upon tumor cell recognition, co-opts the natural T cell receptor (TCR) to yield safer anti-tumor responses versus chimeric antigen receptor (CAR) T cells. In preclinical studies, TAC T cells led to complete tumor clearance in mouse models of human cancer, without any TAC-related toxicities. In the ongoing clinical trial (NCT04727151), TAC01-HER2 treatment of HER2+ solid tumors is hypothesized to result in safe and effective anti-tumor responses. Subjects undergo leukapheresis, bridging therapy (if needed) while their TAC01-HER2 are engineered, lymphodepletion chemotherapy (LDC), and finally TAC01-HER2 infusion. Trial Design and Early Results: In phase I dose escalation, the primary objective is to evaluate the safety of TAC01-HER2 at increasing doses of 0.3, 0.8, 3, and 8 × 106 cells/kg in HER2+ solid tumors (1+, 2+ or 3+ as identified by immunohistochemistry) in adult subjects who have progressed after ≥2 lines of systemic therapy. Dose limiting toxicities (DLTs) are assessed up to 28 days from cell infusion. In Phase II, dose expansion groups will further evaluate the safety, efficacy, and pharmacokinetics of the optimal TAC01-HER2 dose in HER2+ breast and other solid tumor types. As of 12 Dec 2022, 12 patients have been treated in Cohorts 1-4 with breast, colorectal, gall bladder, gastro-esophageal junction, gastric and ovarian tumors. No DLTs or neurotoxicity events have been reported. All patients treated so far at Cohorts 3 and 4 (3 × 106 and 8 × 106 cells/kg, respectively) experienced Grade ≤2 CRS which were resolved with standard of care treatments. Eight subjects have reported a total of 15 serious adverse events, all unrelated to TAC01-HER2 except for a Grade 1 and a Grade 2 CRS, both resolved with standard of care. Most adverse events were related to LDC or the underlying neoplasm. At Cohort 2, a partial response was observed in a subject with refractory metastatic gastric adenocarcinoma (3+ HER2) at 1st scan, with a 36.5% reduction in measurable disease and clinical benefit was maintained for 6 months. A 71% disease control rate was observed at Cohorts 2 and 3 at 1st scan (0.8 × 106 and 3 × 106 cells/kg, respectively)). Dose escalation of TAC01-HER2 is ongoing, with the first subject treated at Cohort 4. These results in a heavily pre-treated cancer population show manageable safety and promising clinical activity with a novel T cell therapy that may have broad clinical applicability in HER+ cancers. Citation Format: Ecaterina Elena Dumbrava, Daniel Olson, Samuel Saibil, Brooke Pieke, Mridula A. George, Riemke Bouvier, Miriam Gavriliuc, Kelly Gruber, Kara Moss, Nathan Ternus, Maria Apostolopoulou, Deyaa Adib, Benjamin L. Schlechter. A phase I/II trial investigating safety and efficacy of autologous TAC01-HER2 in relapsed or refractory solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT234.

Exhausted T cells hijacking the cancer-immunity cycle: Assets and liabilities.

T cell exhaustion is an alternative differentiation path of T cells, sometimes described as a dysfunction. During the last decade, insights of T cell exhaustion acting as a bottle neck in the field of cancer immunotherapy have undoubtedly provoked attention. One of the main drivers of T cell exhaustion is prolonged antigen presentation, a prerequisite in the cancer-immunity cycle. The umbrella term "T cell exhaustion" comprises various stages of T cell functionalities, describing the dynamic, one-way exhaustion process. Together these qualities of T cells at the exhaustion continuum can enable tumor clearance, but if the exhaustion acquired timeframe is exceeded, tumor cells have increased possibilities of escaping immune system surveillance. This could be considered a tipping point where exhausted T cells switch from an asset to a liability. In this review, the contrary role of exhausted T cells is discussed.

Treatment of penoscrotal basal cell carcinoma - an institutional retrospective review.

Basal cell carcinoma (BCC) of the sun-protected genital region is rare. We examined all penoscrotal BCC at a single institution over an 18-year period. A total of 7 cases were identified, 5 scrotal and 2 penile. Four cases (57%) were treated with Mohs micrographic surgery (MMS) and 3 cases (43%) with conventional excision. A prior retrospective review of vulvar BCC at our institution during the same time period showed that MMS was utilized in 4 (11%) of 35 cases. Functional preservation is vital when performing surgery on the genitals and tissue-sparing MMS provides optimal tumor clearance and outcomes for both males and females.

Abstract 2976: A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM

Abstract Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumors in adults with a five-year survival rate less than 5%. In recent years, immunotherapeutic approaches that target a tumor-associated antigen (TAA), including EGFRvIII-targeted cancer vaccine and adoptive cell transfer, have been examined in clinical studies, which so far have shown limited efficacy in GBM patients. GBM is highly heterogeneous for targeting antigen expression. An immunotherapeutic agent that harnesses multiple TAAs simultaneously may mitigate tumor antigen escape and improve efficacy in GBM patients. Here, we describe a DNA-encoded tri-specific T cell engager (DTriTE) that targets CD3 and two TAAs, EGFRvIII and IL13Ra2, which are expressed in up to 30% and 75% of GBM cases, respectively. We designed EGFRvIII-IL13Ra2-DTriTE to include single-chain variable fragments targeting IL13Ra2, CD3, and EGFRvIII in a series, fused with glycine-serine linkers. EGFRvIII-IL13Ra2-DTriTE exhibited robust binding activities to both EGFRvIII+ and IL13Ra2+ tumor cells and primary T cells, inducing robust T cell activation and anti-tumor cytotoxicity. EGFRvIII-IL13Ra2-DTriTE showed durable in vivo expression of over 10 weeks after a single IM injection in NSG mice. We developed an orthotopic challenge of a heterogeneous GBM model, wherein a mixture of EGFRvIII+ tumor cells and IL13Ra2+ tumor cells were planted in the brains of NSG mice and primary human T cells were peripherally delivered. A single IM administration of EGFRvIII-IL13Ra2-DTriTE resulted in complete tumor clearance. An administration of DNA-encoded bispecific T cell engager (DBTE) targeting EGFRvIII alone induced tumor clearance in 3 of 10 (30%) and limited tumor control in 5 of 10 (50%) animals. Similarly, mono-targeting of IL13Ra2 alone induced tumor clearance in 1 of 9 (11%) and limited tumor control in 4 of 9 (44%) animals. EGFRvIII-IL13Ra2-DTriTE treatment exhibited significantly improved survival with 8 of 8 (100%) animals survived, while treatments of single EGFRvIII-DBTE, IL13Ra2-DBTE or a vehicle control resulted in 4 of 10 (40%), 4 of 9 (44%), and 0 of 10 (0%) animals survived, respectively. This study shows that dual tumor-targeting approach using DTriTE is highly effective at treating antigenically heterogeneous GBM tumors. Further study of DTriTE multi-antigen approaches for GBM and other heterogeneous tumors appears important. Citation Format: Daniel Hongil Park, Pratik Bhojnagarwala, Kevin Liaw, Devivasha Bordoloi, Ebony N. Gary, David B. Weiner. A single administration of DNA-encoded tri-specific T cell engager (DTriTE) targeting EGFRvIII, IL13Ra2, and CD3 shows complete tumor clearance in an orthotopic challenge model of heterogeneous GBM [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 2976.

Abstract 899: Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer

Abstract As immune-therapies continue to develop for solid tumors, the tumor microenviroment (TME) poses many challenges that CAR T-cells need to overcome for efficient tumor cell clearance. Tumor immune evasion mechanisms, presence of immunosuppressive factors and cellular resistance to tumor infiltration are key mechanisms that the TME uses to reduce efficacy of T-cells against the tumor. Therapeutic options for triple negative breast cancer (TNBC) remain limited to date despite it being the most aggressive subtype of breast cancers and carrying the poorest prognosis. Tumor-associated MUC1 antigen is overexpressed in a large number of TNBC patients offering an effective discriminatory target for CAR T-cell therapy. Here, we present innovative strategies exploring alternative delivery routes and purposeful armoring of CAR T-cells to boost efficiency while preserving safety. To maintain anti-tumor CAR T-cell cytotoxic and proliferation in the hostile TME of solid tumors, we developed state-of-the-art multiplexed gene editing using high precision TALEN® technology. With that aim, we armored allogeneic CAR T-cells with specific attributes to release immune inflammatory cytokines and protect from inhibitory effects of the TGFB1 and PD1 pathways. A combination of strategies using allogeneic CAR T-cells with diverse attributes were evaluated against TNBC tumors in various in vivo pre-clinical models. Local CAR T-delivery resulted in effective control of tumor growth and robust antigen specific recognition. Furthermore, we assessed how the delivery route contributes to CAR-T cell distribution. CAR T-cell activity could also be increased systemically upon tumor-specific release of immunostimulatory cytokines without compromising safety. Our results demonstrate not only superior activity of armored MUC1-CAR T-cells resulting in tumor clearance but also in the recovery of normal glands. Thus, innovative strategies can be used to allow CAR T-cell efficiency in the hostile tumor microenvironment while preserving safety. Overall, our pre-clinical results underline the capability of armored allogenic MUC1-CAR T-cells to excel in the immune suppressive tumor micro-environment, suggesting that allogenic MUC1-CAR T-cell therapy could be an effective option in treating relapsed/refractory TNBC patients with limited therapeutic options. Citation Format: Piril Erler, Hana Cho, Jordan Skinner, Laurent Poirot, Beatriz Aranda-Orgilles. Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer [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 899.

Abstract 5163: Local delivery of mRNA immunotherapy encoding HPV16 antigen, IL-12, and LIGHT/TNFSF14 results in superior immunogenicity and tumor clearance in a murine model of HPV16-driven cancer

Abstract While intramuscular vaccination with HPV16 antigens has been shown to induce robust tumor-specific immune responses in the periphery, this has led to limited efficacy in the clinic. One potential explanation is the lack of sufficient infiltration of antigen-specific T cells into the tumor. The few T cells that make their way into the tumor are met with a plethora of negative signals that suppress their activity and abrogate local T cell expansion. To overcome this limitation of intramuscular cancer vaccines, we have developed an approach using in situ vaccination with a combination of immune modulators and tumor antigens, all encoded by mRNAs and delivered using nanoparticles. In this study, we show the superior efficacy and immunogenicity of a locally delivered mRNA cancer vaccine over that of an intramuscular HPV16 cancer vaccine. Both routes of administration result in robust induction of antigen-specific T cells, but local administration of mRNA-encoded antigens and immune modulators also resulted in enhanced infiltration of immune cells into the tumor, local Th1/M1 cytokine induction, and more-complete tumor clearance. Specifically, we found that the inclusion of the immunomodulators IL-12 and TNFSF14/LIGHT enabled tumor clearance in a murine tumor model. While tumors treated with mRNA-encoded HPV16 antigen alone induced robust systemic antigen-specific responses, minimal levels of local T cell expansion and infiltration, proinflammatory cytokine induction, and tumor control were observed. By contrast, tumors treated with mRNA-encoded HPV16 antigen, IL-12p70, and TNFSF14 completely cleared large established HPV16 expressing tumors with long-lasting recurrence free survival and immune memory. This observation was replicated in in a second murine tumor model, confirming the superior potency of locally delivered mRNA cancer vaccines in combination with immunomodulators. Consequently, we are developing a locally administrated mRNA therapy for the treatment of patients with cervical intraepithelial neoplasia (CIN). Citation Format: Weiqun Liu, Diane M. Da Silva, Edward E. Lemmens, Colin J. McKinlay, Sangeeta Nath, Meredith L. Leong, Samuel Deutsch, W. Martin Kast, Ole Audun W. Haabeth. Local delivery of mRNA immunotherapy encoding HPV16 antigen, IL-12, and LIGHT/TNFSF14 results in superior immunogenicity and tumor clearance in a murine model of HPV16-driven cancer. [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 5163.

Abstract 1780: TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements

Abstract The clinical success of cancer immunotherapy, including engineered T cell therapies, has revolutionized treatment paradigms and patient outcomes. While hematological tumors have benefited most from cell therapy approaches, the treatment of solid tumors remains a challenge in part due to the limited availability of abundant and tumor-specific cell-surface antigens. Peptides derived from intracellular tumor-specific proteins, such as cancer-testis antigens (CTAs), that are presented via HLA (pHLA) enable a therapeutic opportunity to target tumors while sparing normal tissue. Surface-accessible pHLA complexes may be targeted with engineered T cell receptors or TCR mimetic antibodies reformatted to chimeric antigen receptors (CARs). Using MAGE-A4 as a model CTA, we compared engineered human TCR- and CAR-T cells head-to-head to understand how to best deploy these modalities. To this end, we generated fully-human, HLA-A2/MAGE-A4(230-239)-specific TCR and CARs harboring CD28/CD3z or 41BB/CD3z signaling domains. TCR and CAR-T cells demonstrated similar robust on-target reactivity, cytokine release, and target cell lysis in vitro. In vivo, each of these modalities showed potent, dose-dependent anti-tumor efficacy against human xenograft tumors expressing low, endogenous levels (~500 cell-surface copies) of the MAGE-A4 peptide. However, differences emerged when we examined the in vivo kinetics and durability of tumor suppression. MAGE-A4 CD28/z CAR-T demonstrated the most rapid and potent tumor clearance, while the 41BB/z CAR-T showed delayed but ultimately complete efficacy. TCR-T cells induced tumor regressions during the first 2 weeks of treatment, but this response was transient and followed by tumor relapse. These differential responses correlated with early, modest accumulation of CD28/z CAR-T in line with fast tumor clearance. 41BB/z CAR-T showed a remarkable ~800-fold expansion in the tumor versus limited in vivo TCR-T proliferation. Mechanistically, the MAGE-A4 TCR induced strong CD3 proximal signaling associated with a greater induction of T cell dysfunction markers and limited cytotoxic potential in vitro. However, stimulating 41BB signaling pathways in the MAGE-A4 TCR T cells augmented long-term cytotoxicity. These data demonstrate that tumor-specific pHLA complexes can be potently targeted by both TCR and CAR-T cells, and that co-stimulatory signaling is necessary to mediate durable anti-tumor activity. Citation Format: Corinne E. Decker, Jacqueline Idun, Katja Mohrs, Thomas Meagher, Kevin Bray, Iryna Petriv, Jonathon Golas, Timothy Helms, Dharani Ajithdoss, Gavin Thurston, John Lin, Jessica R. Kirshner, David J. DiLillo. TCR-T and CAR-T cells targeting HLA-A2/MAGEA4 demonstrate differential tumor control, reflecting co-stimulatory signaling requirements [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 1780.

Abstract 408: Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice

Abstract Here we present a new and improved GHR antagonist candidate (compound G) with novel discrete PEGylations exhibiting marked efficacy in synergizing anti-cancer therapies. The American Cancer Society estimates the 5-year survival rates of pancreatic adenocarcinoma (PAAD) and bile duct cancer/cholangiocarcinoma (CHOL) to be a dismal 10% in 2022, indicating an urgent need for new therapeutic approaches for both of these devastating diseases. Several human cancers, including PAAD and CHOL express two validated oncological targets - the growth hormone receptor (GHR) and the insulin-like growth factor 1 receptor (IGF1R), as well as their cognate ligands growth hormone (GH) and insulin-like growth factor 1 (IGF1), respectively. Our laboratory had pioneered the discovery of pharmacologic antagonists for the growth hormone receptor (GHR), including the only approved and prescribed GHR antagonist, pegvisomant (Pfizer), prescribed for acromegaly (a condition of excess GH due to a hypersecreting pituitary adenoma). GHR antagonists not only inhibit the effects of GH but also robustly suppress the production of IGF1. The novel GHR antagonist compound G, that we present here, has a greater binding affinity than Pegvisomant to the GHR. And unlike Pegvisomant, compound G binds to the prolactin receptor as well as the GHR. A large body of research by us and others have earlier shown that the covert actions of tumoral GHR and IGF1R activation collectively drive mechanisms of therapy resistance including, but not limited to, active multidrug efflux, activation of epithelial-to-mesenchymal transition (EMT), resistance to apoptosis, increased fibrosis, increase of cancer stem cells, tumor angiogenesis and senescence. Our novel discrete PEGylated GHR antagonist, compound G, alone exhibits significant suppression of cancer cell viability, invasive potential, and drug efflux rate in PAAD and CHOL cell cultures. In Nude mice with human PAAD or CHOL xenografts, GHR inhibition by compound-G markedly sensitized the tumors towards both low and high doses of gemcitabine (for PAAD) or gemcitabine-cisplatin (for CHOL) treatments. Compound-G in combination with chemotherapy resulted in markedly reduced tumor sizes in all CHOL and PAAD mice and almost complete tumor clearance in 40% of the mice with PAAD. In the GHR antagonist treated tumors, significantly lower expression of ABC transporters (Abcb1, Abcg5, Abcc2, Abcg2), EMT mediators (Cdh2, Zeb1, Snai2, Epas1) and apoptosis marker (Bcl2) genes compared to control were observed - accounting for the molecular mechanisms underlying the observed effects. Collectively, our data provide a robust pre-clinical validation for treatment regimens combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Citation Format: Reetobrata Basu, John J. Kopchick. Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice [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 408.

Abstract 4108: Transient epigenetic reprogramming enhances T-cell proliferation and tumor clearance

Abstract Manufacturing-induced differentiation and accelerated T-cell exhaustion prevent CAR-T therapies from reaching their full therapeutic potential and broader application in the treatment of cancer. To combat these inherent challenges, Turn Biotechnologies is developing its novel and proprietary mRNA-based technology, Epigenetic Reprogramming of Aging (ERATM), to i) preserve a less differentiated status; ii) diminish (or even eliminate) exhaustion; and iii) improve the cytotoxicity and efficacy of CAR-T cell cells. Prolonged CAR-T cell manufacturing process leads to a more differentiated and effector-like phenotype, which is susceptible to accelerated exhaustion in vivo. Unfortunately, clinical evidence suggests that less differentiated and stem-cell like CAR-T products have increased proliferative capacity, reduced exhaustion, and greater efficacy. Additionally, aging is known to compromise T-cell function, reducing chances of CAR-T therapy candidacy for older patients. These compounded effects of manufacturing and aging severely limit CAR-T cell functionality in terms of proliferation, perdurance, and cytotoxic capacity, as well as precluding wider patient access. Leveraging our previous work with other aged human cells, we hypothesized that the transient expression of ERATM factors could impact manufacturing of CAR-T cells by mitigating their differentiation, promoting T stem cell memory (Tscm) phenotype, reducing/eliminating exhaustion, and resetting the epigenetic clock in CAR-T cells. Here, we present preclinical data showing that ERATM treatment of T cells delivered using our proprietary eTurnaTM platform can restore youthful functionality, reducing exhaustion and effects of aging, improving their ability to fight cancer while safely maintaining cellular identity. ERATM -treated T cells have higher proliferation capacity and cytotoxicity upon tumor cell engagement in vitro compared to control cells. Treatment of ERATM -treated T-cells also increases the production of beneficial cytokines to support their survival and cytotoxic activity. Importantly, ERATM treatment preserves T-cell identity and T-cell repertoire, while avoiding unwanted hyperproliferation or clonal expansion. In murine models of hematological cancers, these effects of ERATM treatment on T-cells results in superior cancer cell killing and clearance. These benefits of ERATM technology to current CAR-T manufacturing processes have the potential to translate into the clinic as improved patient outcomes and survival with reduced side effects, thereby expanding patient access and enabling immunotherapies to serve as a first-in-line lifesaving solution. Citation Format: Alec McQuiston, Junbao Yang, Jason Romero, Naveen Bojjireddy, Travis McQuiston, Vittorio Sebastiano, Mustafa Turkoz. Transient epigenetic reprogramming enhances T-cell proliferation and tumor clearance. [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 4108.

Abstract 2911: Identification of novel CAR transmembrane and endodomains for improved persistence and cytotoxicity of NK cell therapies

Abstract Natural killer cells offer a promising treatment strategy for cancer due to their efficacy and favorable safety profile. However, CAR NK cells in solid tumors have had limited success and considerable efforts are being undertaken to improve their efficacy and persistence. Designing optimal CAR transmembrane and intracellular endodomains may increase expression, strength of activation, co-localization within synapses, and improve functional outcomes. First generation NK CARs had CD8ɑ or CD28 transmembrane domains, similar to CAR T cells. They also included endodomain combinations of CD3ζ, CD28, and 4-1BB, while subsequent generations included combinations of NKG2D, DNAM-1, and 2B4 endodomains, with the intent of incorporating NK-specific activation signals. To identify optimal design sequences for NK-based CARs, we developed an in vitro screening platform based on the functional attributes of NK cells, cytotoxicity and persistence. We designed and synthesized two CAR libraries comprising ~2,700 and ~1,300 endodomains drawn from pairwise combinations of 52 and 36 co-stimulatory motifs, respectively. Through a pooled screening experiment, we identified multiple signaling domains that improved the persistence of NK cells in low cytokine conditions throughout two weeks of re-stimulation. Isolated lines of NK cells expressing the top hits demonstrated higher cytotoxicity both immediately after transfection and after two weeks of re-stimulation with tumor cell lines, validating their improved therapeutic potential. We additionally trained a machine learning model using the counts of signaling domain-binding motifs as features to predict the cytotoxicity and persistence of the screened endodomains. This discriminator model and the feature importance can be used to recommend novel co-stimulatory motif combinations for future screens. Top hits in these screens included co-stimulatory domains known to enhance proliferation, cytotoxicity, and tumor clearance in animal models and commonly used in CAR T cell therapies. We frequently observed these domains in novel combinations or in combination with other endodomains not presently used in CAR T therapies. Additionally, we identified transmembrane domains derived from NK surface receptors that increased the presentation of CAR on the NK cell surface. We subsequently integrated these transmembrane domains with hits from our endodomain screen and again used re-stimulation and cytotoxicity assays to functionally test potentially positive interactions between transmembrane domains and endodomains in CAR-NKs. These screening data are ideal for developing predictive models endodomain combinations that enhance NK cell cytotoxicity and persistence. Our results suggest that this approach can be used to identify CAR structural components tailored to cell type, binder specificity, and tumor microenvironment. Citation Format: Maddie Williams, Aye Chen, Matt Stone, Rebekah Turk, Nora Kearns, Nick Bogard, Lan Guo, Brian Belmont, Mary H. Young, Bryce Daines, Max Darnell. Identification of novel CAR transmembrane and endodomains for improved persistence and cytotoxicity of NK cell therapies [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 2911.

Abstract 1150: CD70 HIT T cells overcome tumor antigen heterogeneity in renal cell carcinoma

Abstract CAR T cells have revolutionized the treatment of hematologic malignancies but have not yet achieved the same curative or deep responses for solid tumors like renal cell carcinoma (RCC). One of the major reasons for this lack of clinical success is the heterogeneous expression of target tumor antigens in solid tumors. In order to better understand the limitations of CAR T cell therapy in the solid tumor setting, we utilized two clear cell RCC patient-derived xenograft models (PDX), which replicate CD70 and carbonic anhydrase IX (CAIX) heterogeneity of expression seen in patient kidney tumor samples. We evaluated individual CD70 CAR T or CAIX CAR T cells in PDX-bearing NSG mice and showed only partial tumor clearance. This was not due to lack of tumor infiltration since we observed trafficking to tumor sites with in vivo T cell and tumor imaging. Even with a dual targeting approach combining CD70 and CAIX CAR T cells to increase tumor coverage, we did not eradicate the kidney tumors. By analyzing the tumor antigen profiles of relapsing kidney tumors, we found that the CD70 and CAIX positive populations were cleared but detected a residual CD70 and CAIX double-negative population. Based on transcriptomic and proteomic analyses of these residual populations, we detected CD70 expression, which suggested that these CD70/CAIX double negative populations did in fact, have ultra-low CD70 expression which was not at a level high enough to be eliminated by CD70 CAR T cells. This key finding revealed that CD70 expression was not present or absent but rather present in a spectrum ranging from high to very low. Based on this fundamental observation, we hypothesized that a highly sensitive HLA-independent T cell receptor (HIT receptor) may be used to target this ultra-low CD70 population. Indeed, treatment with CD70 HIT T cells resulted in complete and long-lasting tumor cures in both PDX models. Our findings show that we can overcome the major challenge of tumor antigen heterogeneity in RCC because apparent CD70-negative tumor populations retain CD70 expression and can be targeted by a highly sensitive HIT T cell. This represents a significant advance for the development of a potentially effective cellular therapy for RCC, which may be applicable to other aggressive CD70-positive tumors like ovarian and pancreatic cancers. Our findings underscore the critical importance of assessing antigen expression in a quantitative manner to determine if antigens are truly absent or present at very low levels, in which case they may be responsive to an engineered antigen-sensitive immune cell as afforded by the HIT receptor. Citation Format: Sophie A. Hanina, Michael Lopez, Huiyong Zhao, Rajasekhar Vinagolu, Ritesh Kotecha, A Ari Hakimi, John H. Healey, Jorge Mansilla-Soto, Michel Sadelain. CD70 HIT T cells overcome tumor antigen heterogeneity in renal cell 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 1150.

Abstract 3239: Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic cancers

Abstract Background: PD-1 blockade (αPD-1) has shown limited efficacy in breast & gynecologic cancers. In other types of solid tumor, regulatory T cells (Tregs) harbouring markers of highly suppressive effector Tregs (eTregs) correlates with poor responses to PD-1 blockade, prompting combination therapy based on eTregs depletion. Yet, subsets of Tregs remain to be determined in breast & gynecologic cancers, in order to: (i) evaluate the role of eTregs in resistances to PD-1 blockade, (ii) identify the most selective target for eTregs depletion tailored to tumors context and combination strategy. Methods: We collected public single-cell RNA/TCR-sequencing data from primary tumors and metastases of Triple-Negative Breast Cancer patients (TNBC, N = 28) biopsied before and after αPD-1 (pembrolizumab). Microarray data from primary TNBC (N = 124) biopsied before and after αPD-1 were quired for association with clinical responses. Deep phenotyping of Tregs from normal breast tissues (N = 4) and primary breast tumors (N = 8) or gynecologic tumors (N = 17) was performed by mass cytometry. ALD2510 (αCD25NIB, Alderaan Biotechnology) is a novel non-IL-2 blocking Fc-optimized anti-CD25 mAb (1). αPD-1 and αCD25NIB combination was evaluated using: (i) a humanized αCD25NIB in CD34+ humanized NSG mice grafted with human TNBC cell lines, (ii) a murine surrogate of αCD25NIB in a syngeneic TNBC mouse model. Results: CD25high Tregs were accumulating in breast & gynecologic tumor tissues. In TNBC patients treated with αPD-1, fractions of CD25high Tregs were further increased. CD25high Tregs highly expressed eTregs-related molecules in primary tumors, invaded tumor-draining lymph nodes and distant metastases. Amongst the potential therapeutic targets for Treg depletion, only CD25, 4-1BB and CCR8 were largely restricted to intratumoral CD25high eTregs. Yet, CD25 was the best candidate for CD25high eTregs depletion with limited on-target off-Treg effects, as: (i) CCR8 marked a small fractions of CD25high eTregs in primary tumors and was not detected in metastasis, (ii) 4-1BB marked antigen-experienced αβCD8 T cell revigorated by PD-1 blockade, (iii) only CD25high eTregs expressed 4-1BB and CCR8, yet TCR clonotype analysis revealed that CD25high eTregs originate in part from activated CD25+ Tregs, suggesting that anti-CCR8/-4-1BB mAbs may be ineffective due to CD25high eTregs replenishment in tumors. In murine models resistant to αPD-1 alone, αCD25NIB effectively depleted intratumoral CD25high eTregs, with limited effects on peripheral Tregs, and synergized with αPD-1 by restoring a positive effector αβCD8 T cells/Tregs ratio, leading to tumor clearance without adverse effects. Conclusions: This study supports clinical evaluation of CD25high effector Tregs depletion by ALD2510 in patients with breast or gynecologic cancers resistant to PD-1 blockade. Citation Format: Stéphane FATTORI, Aude Le Roy, Jemila Houacine, Lucie Robert, Riad Abes, Laurent Gorvel, Samuel Granjeaud, Marie-Sarah Rouvière, Amira Ben Amara, Nicolas Boucherit, Carole Tarpin, Jihane Pakradouni, Emmanuelle Charafe-Jauffret, Julien Barrou, Gilles Houvenaeghel, Eric Lambaudie, François Bertucci, Philippe Rochigneux, Anthony Gonçalves, Arnaud Foussat, Anne-Sophie Chrétien, Daniel Olive. Selective depletion of regulatory T cells by ALD2510, a novel IL-2-sparing anti-CD25 antibody, synergizes with PD-1 blockade in breast and gynecologic 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 3239.

Abstract 5747: A novel combination therapy of arenavirus vectors and PD1-IL2v strongly potentiates tumor specific T cell responses resulting in synergistic anti-tumor efficacy

Abstract It is undisputed that CD8 T cells play a critical role in controlling tumor growth and killing tumor cells, and that tumor specific T cells are essential for clinical responses to immune checkpoint inhibitors. Yet, the induction and maintenance of functional tumor antigen specific T cell responses represent still one of the greatest challenges in cancer immunotherapy. Therapeutic cancer vaccines based on engineered arenavirus vectors have been shown preclinically to overcome self-tolerance to tumor associated antigens (TAA) and mount unprecedented T cell responses, both, in preclinical tumor models as well as HPV16+ cancer patients in an ongoing Phase 1/2 trial. The novel PD-1 targeted IL-2 variant (PD1-IL2v) was created to maximize the potency of IL-2R activation of effector T cells while overcoming the toxicities of wildtype IL-2. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell was recently shown to promote differentiation of stem-like CD8 T cells into better effectors and was therefore identified as an ideal compound for combination therapies with arenavirus vectors. Combination of vectors encoding tumor associated foreign or self-antigens with PD1-IL2v lead to a massive, up to 60-fold, increase of peripheral tumor specific CD8 T cell numbers compared to vector treatment alone. Notably, frequency of peripheral regulatory T cells was not enhanced by this combination. The massive increase of TAA specific CD8 T cells was associated with synergistic anti-tumor efficacy in two independent tumor models, resulting in longer survival times and up to 60% complete responders. Complete tumor clearance resulted in a stable memory T cell population and protection against tumor re-challenge. Ongoing studies will address modifications of the tumor microenvironment and further characterization of tumor infiltrating T cells upon combination therapy. These preclinical data establish the combination of arenavirus vectors and PD1-IL2v as a promising next generation cancer immunotherapy. Citation Format: Judith Strauss, Diana Reckendorfer, Kimberly Pojar, Laura Codarri Deak, Valeria Nicolini, Roger Sutmuller, Christian Klein, Pablo Umaña, Klaus K. Orlinger, Henning Lauterbach. A novel combination therapy of arenavirus vectors and PD1-IL2v strongly potentiates tumor specific T cell responses resulting in synergistic anti-tumor efficacy. [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 5747.

Abstract 6362: Overcoming resistance to immunotherapy due to loss of antigen presentation

Abstract Resistance to immune checkpoint inhibitors represents a major therapeutic challenge, as only 40% of patients with melanoma (and less with other tumor types) have a long-term response to anti-PD-1 therapy. Resistance can arise because of somatic mutations in cancer cells that allow evasion of T cell-mediated killing. One commonly acquired resistance mutation in melanoma, loss of beta-2 microglobulin (B2m), prevents T cell killing by hiding cancer cells from CD8+ T cell recognition. To understand the failed immune response against resistant tumors, we used single-cell RNA-seq to characterize tumor-infiltrating immune cells in antigen presentation-deficient human melanoma biopsies and CRISPR-modified mouse melanoma tumors. Our data demonstrate an increase in immunosuppressive M2-like macrophages and absence of CD8+ T cells in B2m-null tumors. To overcome this resistance, we treated tumor-bearing mice with CD40 agonist antibody, which promotes differentiation of macrophages towards a pro-inflammatory phenotype and increases dendritic cell priming of CD8+ T cells. Treatment with CD40 agonist reduced tumor growth and improved tumor clearance in B2m-null melanoma and colorectal cancer models. To determine how CD40 agonist treatment works, we depleted different immune populations from the tumor microenvironment. We hypothesized that by depleting M2 macrophages, CD40 agonist treatment would remove an immunosuppressive brake to allow natural killer (NK) cells to kill tumor cells lacking MHC expression. To our surprise, NK cells were not required for the efficacy of CD40 agonist. Instead CD8+ T cells were required, even though the CD8+ T cells cannot directly recognize the tumor cells. scRNA-seq identified a transcriptionally unique state of CD8+ T cells that is recruited to the tumor microenvironment after CD40 agonist treatment. These CD8+ T cells produce IFNγ, which is required for the efficacy of CD40 agonist treatment. These data demonstrate that CD8+ T cells, a key mediator of anti-tumor immunity, can still be recruited to control tumors deficient in antigen presentation. More broadly, they suggest that strategies to activate CD8+ T cells may be effective even in the context of acquired resistance to checkpoint inhibitor therapy. Citation Format: Brian C. Miller, Yacine Choutri, Rose Al Abosy, Amy Huang, Emily K. Cox, Matthew P. Zimmerman, Wan Lin Chong, Katherine J. Vietor, Jenna Collier, Sarah A. Weiss, Debattama Sen, W. Nicholas Haining, Arlene H. Sharpe. Overcoming resistance to immunotherapy due to loss of antigen presentation [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 6362.

Abstract 2735: Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome

Abstract Background: We have developed a novel gene therapy platform based on fusogen biology that allows targeted delivery of CAR transgenes to “resting” T cells through systemic administration of a CD8-targeted paramyxovirus-based viral vector (VV). Therapies utilizing autologous CAR T cells have demonstrated success using the murine CD19-specific scFv, FMC63. These murine sequences have the potential to incite T cell and antibody responses against the CAR, which may play a role in CAR T cell elimination. This scenario increases in likelihood in the context of in vivo CAR delivery, where immunosuppression via lymphodepletion is not required. To reduce the potential for CAR-specific immunogenicity, we developed a novel, fully human CD19-specific CAR for use with our in vivo delivery platform. Methods: For efficacy-based screening of human CD19 binders, healthy donor T cells were transduced with VSV-g pseudotyped lentivirus containing second-generation fully human CD19 CARs. FMC63 CARs were produced in a similar fashion and used as benchmark controls for initial characterization studies. CD8-targeted fusogen was generated through targeted mutation to ablate binding to its native receptor. This “blinded” fusogen was subsequently engineered to display a novel scFv that is specific for human CD8α. VV encoding either FMC63 or human CD19 CARs were pseudotyped with CD8-specific fusogen to produce CD8-targeted fusosomes. CD19 CAR efficacy was analyzed in vitro via luciferase assays against NALM-6 or CD19KO NALM-6 tumor lines. Systemic NALM-6 in vivo tumor models (IV injection) were performed in NSG mice. Results: Here we show, our fully human CD19 CAR is comparable to FMC63 CAR in vitro using both short-term luciferase cytotoxicity and long-term Incucyte tumor killing assays. Furthermore, human CD19 CAR T cells demonstrate similar levels of tumor control NALM-6 tumor bearing mice treated with ex vivo generated CD19 CAR+ cells (Mean AUC [n = 3 donors]: FMC63 = 1.26e4, huCD19 = 4.86e4). Our lead human CD19 CAR, like FMC63, lacked killing and cytokine production when cultured with CD19KO NALM-6 tumor cells, demonstrating CD19 antigen specificity. Furthermore, CD8-targeted in vivo delivery of our fully human CD19 CAR showed reduction in NALM-6 tumor load similar to FMC63 CAR (Mean AUC [n = 2 donors]: FMC63 = 1.59e5, huCD19 = 5.06e4). Summary: In vivo delivery of our fully human CD19 CAR with CD8-targeted fusosomes affords tumor clearance comparable to FMC63-CAR in mice bearing NALM-6 tumors. We believe this fully human CD19 CAR has the potential to reduce the risk of immunogenicity compared to a CD19 CAR with the murine scFv FMC63 potentially allowing increased expansion and persistence of CD19 CAR T cells following in vivo delivery in patients. Citation Format: Jeremy M. Kinder, Christie Ciarlo, Chanel Athena Estrada, Neal van Hoeven, Vandana Chaturvedi, Garrett Zipp, Aaron Lampano, Adam J. Johnson, Kutlu Elpek, Terry J. Fry, Aaron E. Foster. Development of a novel, fully human anti-CD19 chimeric antigen receptor for in vivo delivery via CD8-targeted fusosome [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 2735.

Abstract 3188: Patient-derived TAC01-HER2 TAC T cells produced in Cocoon® Platform is highly functional in models of solid tumors

Abstract Background: The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the re-direction of T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the goal to elicit a safe and durable anti-tumor response. TAC01-HER2, a first-in-class TAC T product targeting HER2 (ERBB2), has entered a Phase I/II clinical trial in patients with HER2-positive solid tumors. Here, we characterized the TAC T cell phenotypes and anti-tumor activity of TAC01-HER2 manufactured using leukocytes from Phase I/II patients in nonclinical in vitro and in vivo assays. Materials and Methods: TAC T cell proliferation, activation, and phenotype of patient-derived TAC01-HER2 were assessed by flow cytometric analysis. In vitro anti-tumor cytotoxicity was assessed via a real-time microscopy-based co-culture assay, and in vivo anti-tumor activity of TAC01-HER2 was assessed in mice engrafted with established solid HER2-expressing human tumors. Results: Complex phenotype analysis showed that patient-derived TAC01-HER2 products consisted of a high percentage of memory T cells similar to products generated from healthy donors. Patient-derived products had significant proportions of CD4 and CD8 T cells, with CD4 being the predominant population in several of these. In a 5-day in vitro potency assay, patient-derived products showed effective tumor cell killing at low E:T rations (1:1 to 1:20) which was comparable to product generated from healthy donors. Similarly, intravenous administration of patient-derived TAC01-HER2 in mice carrying HER2-positive tumors xenografts led to a complete and sustained tumor clearance. Conclusion: The in vitro and in vivo data confirm the potency of patient-derived TAC01-HER2 against HER2-expressing solid tumor models. This work combined with other biomarkers may help correlate nonclinical potency with clinical outcomes. Citation Format: Ling Wang, Stacey X. Xu, Tania Benatar, Ritu R. Randhawa, Philbert Ip, Prabha Lal, Thanyashanthi Nitya-Nootan, Laura Ravensbergen, Heather MacGregor, Suzy Prosser, Sadhak Sengupta, Christopher W. Helsen, Andreas G. Bader. Patient-derived TAC01-HER2 TAC T cells produced in Cocoon® Platform is highly functional in models of solid tumors [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 3188.

Abstract 5746: Flexible tumor-specific cytokine and antigen delivery through the T-SIGn vector platform enables effective CAR-T cell therapy against solid tumors

Abstract Efficacy of Chimeric Antigen Receptor (CAR) T cell therapy against solid tumors is hampered by multiple barriers, including tumor-associated immunosuppression, poor CAR-T cell trafficking into solid tumor masses and shortage of highly expressed cancer-specific target antigens. The T-SIGn (Tumor Specific ImmunoGene) platform generates viral vectors that can produce combinations of transgenes selectively within the tumor microenvironment (TME). T-SIGn is clinically validated for intravenous (i.v.) delivery, enabling vectors to reach primary and metastatic sites to produce their therapeutic payloads specifically in malignant epithelial cells. Using an A549 human tumor xenograft and metastasis model, we previously demonstrated that i.v. administration of a T-SIGn vector encoding IFNα, MIP1α and CD80 results into potentiated CAR-T cell activation and therapy efficacy (Sonzogni et al. 2022). Here, we further evaluated the potential of T-SIGn combination with Cell Therapy by investigating the impact on CAR-T cell activity of vectors encoding different arrays of cytokines and chemokines. Using an in vivo A549 model and anti-HER2 CAR-T cells in NSG mice, we demonstrated that T-SIGn vectors can enable effective CAR-T cell therapy against solid tumors by providing diverse localized cytokine/chemokine-mediated T cell boosting, with IL-12-encoding vectors resulting in complete and durable tumor clearance. Furthermore, we explored the use of the T-SIGn platform to encode secreted bispecific “adaptor” molecules able to simultaneously bind to a tumor surface antigen and to a CAR specific for an antigen not naturally expressed by the tumor, allowing the redirection of CAR-T cells against desired tumor types. We designed a vector (NG-1125) encoding an anti-HER2 ScFv_CD19 bispecific construct as a model “adaptor” molecule, together with IFNα and CXCL9, and we showed that this construct can effectively redirect anti-CD19 CAR-T cell against HER2+CD19− tumor cells. In vitro X-CELLigence-based cytotoxicity assays demonstrated killing of both HER2+CD19− A549 and SKOV3 cells by anti-CD19 CAR-T cells in presence of vector-derived anti-HER2 ScFv_CD19. Following i.v. NG-1125 dosing of A549 tumor-bearing mice, ex vivo flow cytometry analysis of tumor masses revealed binding of the encoded adaptor molecule to both vector-infected and uninfected tumor cells. Furthermore, in vivo NG-1125 administration together with anti-CD19 CAR-T cells resulted into increased intra-tumoral densities of transferred T cells. Collectively, these data show the potential of T-SIGn vectors in overcoming solid tumor resistance mechanisms to CAR-T and pave the way for the development of other immunotherapeutics to be expressed within the TME via T-SIGn vectors in combination with CAR or TCR-based therapies. Citation Format: Maria Stella Sasso, Rachel Bergin, Rochelle Lear, Darren Plumb, Manuela Zonca, Eva Vainiute, Meg Snowden, Tae Hyun Jang, Alice Muntzer, Carla C. Cerqueira, Katy West, Samantha Bucktrout, Brian R. Champion. Flexible tumor-specific cytokine and antigen delivery through the T-SIGn vector platform enables effective CAR-T cell therapy against solid tumors. [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 5746.

Abstract 6776: Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC

Abstract Background: While cancer therapies are becoming increasingly more targeted, our understanding of the genomic changes occurring during these treatments is lacking. Moreover, the tumoral heterogeneity and its spatial distribution remain important obstacles in predicting tumor evolution and treatment responses. In recent years, genomic spatial profiling techniques have shed light on this complexity by detecting gene expression changes in heterogenous tumor regions and their microenvironment over the course of treatment. Methods: Tepotinib is a highly selective and potent MET class Ib inhibitor, approved for the treatment of patients with NSCLC with MET exon 14 skipping alterations. To detect genomic changes during neoadjuvant treatment with tepotinib, a GeoMx® digital spatial profiler (DSP; Nanostring Technologies) analysis was conducted in paired baseline and on-treatment patient samples. GeoMx® DSP was performed by selecting regions of interest with nuclear, TTF-1, CD3 and CD33 morphology markers followed by next-generation sequencing (NGS) using the Cancer Transcriptome Atlas RNA panel (>1,800 cancer-related genes). Results: Following a poor response to chemotherapy, neoadjuvant tepotinib treatment was initiated in a 54-year-old female, never-smoker with NSCLC harboring a MET exon 14 alteration. Owing to a good response to tepotinib, surgical resection of the tumor was decided by the tumor board given the downgrading (from IIIB to IA1) and resulting in a major pathological response. This resection allowed spatial transcriptomics to be compared to a baseline biopsy from the same patient. MET signaling inhibition by tepotinib treatment increased immune cell infiltration in the tumor microenvironment compared to the baseline sample. This enhanced tumoral immune cell density was shown by the fluorescent morphology markers and confirmed through spatial deconvolution of the NGS results. It was further characterized by the significantly altered gene expression levels between the two samples. Gene ontology (GO) assessment of the differentially expressed genes revealed GO terms associated with tumorigenesis in the basal sample, such as cell mobility and motility, vascular epithelial growth factor production and cell communication. In turn, the tepotinib-treated tissue mainly exhibited tumor-clearing characteristics, including antigen processing and presentation through major histocompatibility complex classes I & II, T cell-mediated toxicity, and granulocyte and leukocyte activation. Conclusion: This is the first spatial genomic characterization of the effect of tepotinib on the tumor and its microenvironment in a patient with NSCLC over time. Results from this case report highlight the impact of MET inhibition by tepotinib on the tumor immune microenvironment, likely enabling the recruitment of antigen-presenting cells for tumor clearance, while reducing the expression of tumor-promoting genes. Citation Format: Manon A. Simard, Felix Geist, Carlos Cabrera, Santiago Viteri, Joachim Albers, Michael Zuehlsdorf, Niki Karachaliou. Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC. [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 6776.

Assessing the Efficacy of Imiquimod Use in Patients With Persistent Locally Advanced Melanoma In Sit.

The standard of care when treating melanoma in situ (MMIS) is an excision with at least 5 mm surgical margins.1 Some studies have suggested up to 9 mm margins to maximize local recurrence-free survival.2 This retrospective review aims to assess the efficacy of imiquimod as a topical treatment for persistently positive MMIS at the margins of prior excisions or where surgery is not an option. Retrospective study conducted at Moffitt Cancer Center between 2019 and 2021 with patients aged > 18 years with MMIS at the margins of excision of an invasive melanoma or MMIS. Included patients were not ideal candidates for primary or additional surgical resection due to non-feasibility of surgery because of comorbidity or cosmetically sensitive location and/or the need for repeated skin grafting, or due to patient's refusal. Patients received imiquimod on protocol for 16 weeks and were monitored for treatment response and side effects. Following completion of the treatment, scouting biopsies were performed to assess histological response, and dermoscopy was used to determine the clinical disease status. Ten patients completed 16 weeks of imiquimod. Seven (75%) had a median of 2 surgical resections, and 3 refused surgery despite discussion that surgery was standard of care. Seven were deemed free of disease on post-imiquimod treatment scouting biopsies, while 2 were found to be clinically free of disease following confocal microscopy, indicating a tumor clearance rate of 90% with imiquimod treatment. One patient was found to have persistent residual disease following 2 rounds of imiquimod and was taken for an additional surgical excision after which they were deemed free of disease. Median follow-up duration from the onset of imiquimod therapy to the last clinic visit was 18 months, without any recurrences to date. Imiquimod appears to demonstrate an encouraging tumor clearance among patients with persistent MMIS after surgery where further surgical resection may not be feasible. Although long-term durability has not been demonstrated in this study, a 90% tumor clearance rate is promising. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.6987.