Heterogeneity Of Antigen Expression Research Articles

Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death.

Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands. In this study, we have investigated the potential for protein-based octomeric chemically self-assembled nanorings (CSANs) to be used for anticancer drug delivery. The CSANs are composed of a DHFR-DHFR fusion protein incorporating an EGFR-targeting fibronectin and the anticancer drug MMAE conjugated through a C-terminal farnesyl azide. The anti-EGFR-MMAE CSANs were shown to undergo rapid internalization and have potent cytotoxicity to cancer cells across a 9000-fold difference in EGFR expression. In addition, anti-EGFR-MMAE CSANs were shown to induce immunological cell death. Thus, multivalent and modular CSANs are a potential alternative anticancer drug delivery platform with the capability of targeting tumor cells with heterogeneous antigen expression while activating the anticancer immune response.

Cotargeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma

AbstractThe remarkable efficacy of Epstein-Barr virus (EBV)-specific T cells for the treatment of posttransplant lymphomas has not been reproduced for EBV-positive (EBV+) malignancies outside the transplant setting. This is because of, in part, the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely LMPs 1 and 2, EBNA1, and BARF1 (type-2 [T2] latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies and, because several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin lymphoma biopsies. This provided the rationale for broadening the target antigen–specific repertoire of EBV-specific T cells (EBVSTs) for therapy. We stimulated, with both lytic and latent cycle proteins, peripheral blood mononuclear cells from healthy donors and patients with EBV+ lymphoma to produce broad repertoire (BR) EBVSTs. Compared with T2 antigen-specific EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NSG mice and produced higher levels of proinflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV+ lymphoma (ClinicalTrials.gov identifier: NCT01555892 and NCT04664179), as well as for other EBV-associated malignancies.

Abstract 5092: Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors

Abstract Clinical success of antigen-directed therapies in cancer has been limited by heterogeneous antigen expression and a narrow therapeutic window. In many patients, failure of antigen-directed therapies such as CAR T-cell therapies, antibody-drug conjugates (ADC) or radioligand therapies (RLT) has less to do with a lack of potency or specificity of the agent and more to do with variable and suboptimal antigen expression in the patient. We are developing combination therapies that introduce a priming agent designed to optimize antigen expression in the patient and improve the therapeutic window of existing antigen-directed therapies. Our high-throughput platform utilizes machine learning and automation to scale functional combinatorial screening for small molecule priming agents using cell lines and patient samples. Using this platform, we are identifying priming therapies for CD123, CD33, CD22, and CD19 in hematological malignancies including acute myeloid leukemia (AML), and HER2, HER3, and TROP2 in solid tumors including breast cancer.For our CD123 program, we have identified priming agents that selectively upregulate CD123 expression ≥2-fold on AML cells without increasing expression on normal myeloid progenitor cells, which also express CD123. AML cells treated with a CD123 priming agent in combination with a CD123 ADC showed greater tumor-cell killing compared to the ADC alone. In comparison, normal myeloid progenitor cells from healthy bone marrow treated with the same CD123 priming-ADC combination treatment did not show an increase in ADC-mediated cell killing. From our mechanistic studies, we have identified a somatic mutation associated with CD123 upregulation that may lead to selective upregulation on cancer cells and not on normal cells. Additionally, this marker can be used for patient selection. We have also observed CD123 upregulation in vivo using mouse models of AML and are currently conducting additional in vivo proof of concept studies to demonstrate that CD123 priming therapy enhances efficacy.There are two distinct advantages of a priming approach paired with an antigen-directed therapy, which include: (1) improving the durability of patient responses and (2) expanding the patient population. Our priming combination approach has the potential to overcome antigen heterogeneity and thereby improve the clinical success of antigen-directed therapies. Citation Format: Kathryn E. Vanderlaag, Anukriti Dhar, Molly Volkman, Erik Werner, Transon Nguyen, Kamran Ali. Development of a priming approach to enhance antigen-directed therapies in hematological malignancies and solid tumors [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 5092.

Abstract 9: AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers

Abstract The KRASG12D mutation is an ideal target for anti-cancer therapies as its expression is typically clonal, restricted to cancer tissue, and is among the most common oncogenic drivers in solid tumors. TCR-T cell therapies have demonstrated clinical activity in some solid cancers but have been limited by heterogeneous antigen expression and unfavorable tumor microenvironments. By targeting the KRASG12D mutation for which the cancer has established genetic dependency, AFNT-212 is designed to selectively target all cancer cells while avoiding on-target/off-tumor toxicities. AFNT-212 is non-virally engineered to knock-in a 5-transgene cassette expressing a high-avidity TCR specific for the KRASG12D mutation, a CD8α/β coreceptor, and a chimeric cytokine receptor. Transgene insertion at the TRAC locus disrupts expression of the endogenous TCRα, further enhancing the expression/activity of the transgenic KRASG12D TCR. Primary human CD8+ and CD4+ T cells were genetically engineered by a novel CRISPR-Cas nuclease system to integrate AFNT-212 transgenes within the TRAC locus. A cGMP compatible scale-up process for non-viral knock-in was established to support AFNT-212 clinical manufacturing. The activity of AFNT-212 was assessed against a panel of human KRASG12D tumor cell lines in vitro and established mouse xenograft models in vivo. The preclinical safety profile of AFNT-212 was evaluated by X-scan and crossreactivity assessment, alloreactivity studies, and cytokine independent growth studies. The specificity of gene-editing (GE) was assessed by an unbiased oligo-capture method followed by targeted sequencing. AFNT-212 TCR-T cells demonstrated potent in vitro anti-tumor activity against endogenously expressing HLA-A*11:01 KRASG12D tumor cells, including during chronic exposure to viable tumor cells. AFNT-212 TCR-T cells showed robust antitumor activity in established xenograft mouse models in vivo. No cross-reactivity was identified for the KRASG12D TCR against potential self-peptides even at supraphysiological levels, demonstrating high specificity of the TCR. No alloreactivity or cytokine-independent proliferation was observed. GE safety evaluations did not reveal any off-target activity using high sensitivity (~0.1%) NGS-based analyses or any GE-associated chromosomal rearrangements. The manufacturing of AFNT-212 consistently delivered >50-fold expansion of engineered TCR-T cells to meet expected clinical dose levels and exhibit memory/stemness phenotypes and negligible markers of immunologic exhaustion. AFNT-212, a novel TCR T cell therapy targeting KRASG12D mutant tumors, demonstrates robust activity against KRASG12D mutant tumors in vitro and in vivo. The robust manufacturing process developed using non-viral gene editing in the TRAC locus will support future clinical development of AFNT-212. Citation Format: Allison Drain, Nicholas Rouillard, Nathaniel Swanson, Martina Canestraro, Santosh Narayan, Tyler Warner, Nicole Danek, Ken Gareau, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Iqraa Priyata, Sarah Vidyasagar, Taylor Riggins-Walker, Hui-Wen Liu, Klaus Pechhold, Lauren Brown, Joshua Francis, Xingyue He, Patrick Browne, Rebecca Lamothe, Meghan Storlie, Gregory Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Christopher A. Klebanoff, Hubert Lam, Ankit Gupta, Damien Hallet, Gary Shapiro, Kim Nguyen, Loïc Vincent. AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers [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 9.

Applicability limitations and mismatched populations of chimeric antigen receptor T cell immunotherapy

In the past ten years, the research have witnessed the emergence of new tumor immune precision targeted therapy CAR-T cells, which has opened up a new path for tumor treatment. CAR-T cell therapy has been approved for the treatment of hematological tumors, but its role in solid tumors is still unsatisfactory, so many studies have explored combination therapies to improve the efficacy of CAR-T therapy in solid tumors, and although CAR-T cell therapy has a good effect on the treatment of hematologic tumors, there are still non-expressed cases.3 Combination immunomodulators. Chimeric antigen receptor T cell therapy is a new type of immunotherapy in the field of tumors, and CAR-T therapy for solid tumors faces many challenges due to the immunosuppressive microenvironment of solid tumors and the heterogeneity of antigen expression. This paper mainly focuses on the range of treated people of chimeric receptor T cells, and roughly summarizes some of the suitable populations through a large number of literature readings and fusion citations, which aims to help more accurately locate the treated population.

Rational Combinatorial Targeting By Adapter CAR T Cells (AdCAR-T) Prevents Antigen Escape in Acute Myeloid Leukemia (AML)

Targeting AML by chimeric antigen receptor T cells (CAR-T) has been challenging due to promiscuous expression of AML-associated antigens on healthy hematopoietic stem and progenitor cells as well as a high degree of inter- and intratumoral heterogeneity. Consequently, we did not yet see the dramatic clinical successes as in B-phenotypic malignancies treated with CD19 or BCMA directed CAR-T. In the present study, we analyzed inter- and intratumoral heterogeneity of AML-associated antigens in 30 primary pediatric AML samples at single cell resolution. We identified CD33, CD38, CD371, IL1RAP and CD123 to be most frequently expressed. Notably, high variability was not only observed across the different patient samples, but also among leukemic cells of the same patient, which indicates that multiplexed targeting approaches will be required to target all leukemic cells. To address this need, we utilized our modular Adapter CAR (AdCAR) platform enabling precise qualitative and quantitative control over CAR-T function. We show highly efficient and target specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. Importantly, we prove that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to mono-targeted CAR-T therapy. For the first time, we demonstrate in a PDX model that rational combinatorial targeting by AdCAR-T can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting, utilizing the AdCAR platform.

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Introduction: Chimeric Antigen Receptor (CAR) T-cell therapies are remarkably efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy B- and plasma-cell counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), as the CAR T-cell activity might lead to terminal ablation of hematopoiesis. In principle, CAR T-cells could be used to abrogate both, AML cells as well as HSPCs, followed by HSPC transplantation, ensuring hematopoietic recovery after termination of CAR T-cell activity and possibly without genotoxic preconditioning. This approach could be particularly beneficial for patients unable to tolerate radiation and high-dose chemotherapy. In order to address this challenge, we developed adaptor-mediated CAR T-cells displaying the single-chain variable fragment E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM). Such tagged adaptors, used as single linkers or in combination, would then act as safety switch, modulating CAR T-cell activation, proliferation, and lytic activity. Results: We generated second-generation AdFITC(E2)-CAR T-cells and multiple diabody-based adaptors against selected AML antigens. We then tested the diabodies targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination. Combinatorial staining with CD117 Db-FM and CD33 Db-FM, each added at saturating concentration for the cognate antigen, resulted in higher fluorescein decoration of target cells, with more than two-fold increased fluorescence intensity detected by flow cytometry upon combined staining, thus enhancing AdFITC(E2)-CAR T-cell target density. In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117 +GFP +Luc +) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to equimolar concentration of single adaptors. To further investigate the basis for increased killing by dual targeting, we performed live-cell imaging of effector-target cell interactions in microwells. The addition of both CD117 and CD33 Db-FM to the co-culture of MOLM14-CD117 +GFP +Luc + and AdFITC(E2)-CAR T-cells resulted in faster tumor cell lysis (measured by influx of PI) which correlated with a more rapid, durable engagement time of CAR T-cells with target cells. We next tested CAR T-cell biocidal activity in therapeutic xenogeneic mouse models engrafted with MOLM14-CD117 +GFP +Luc + where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood as shown by bioluminescence and terminal flow cytometry analysis (Figure 1). Conclusions: We here tested an adaptor CAR T-cell approach using multiple adaptors to on-off modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model with high clinical need for therapeutic improvement. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy, individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight (~55kDa), diabody-based adaptors are rapidly cleared from the body via the kidneys allowing for rapid control over AdFITC-CAR T-cell on-off activity prior to HSPC transplantation. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity towards target cells, while ameliorating the side-effects against healthy tissues expressing only one antigen of the combination.

CCR1-Targeting CAR T Cells for Acute Myeloid Leukemia

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is highly effective in patients with B cell malignancies and multiple myeloma, but initial clinical trials with CARs targeting acute myeloid leukemia (AML) have shown less successful results. Challenges inherent to using CAR T cells for AML include lack of a leukemia-specific target antigen due to heterogeneous antigen expression and potential on-target/off-tumor toxicity due to co-expression of target antigens on normal myeloid cells. C-C chemokine receptor 1 (CCR1, also known as CD191), a G protein-coupled receptor that binds to members of the C-C chemokine family, is a promising AML-associated antigen that is reported to be expressed on over 75% of AML samples and minimally expressed on hematopoietic stem/progenitor cells and T cells. The present study aimed to explore the potential of CCR1 as a novel CAR target antigen using a syngeneic mouse model that allows investigation of both efficacy and toxicity of the CAR T cells. METHODS AND RESULTS We developed a CAR based on the single chain variable fragment (scFv) KM5907 targeting both mouse and human CCR1. We constructed a second-generation CCR1-CAR containing a CD28 hinge, a CD28 transmembrane domain, a CD3 zeta signaling domain, and a CD28 co-stimulatory domain. We mutated the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 zeta chain to retain only a single active membrane-proximal (1XX) ITAM, based on a prior study showing that the 1XX ITAM format enhanced CAR T cell activity by reducing T cell exhaustion. Primary murine T cells were retrovirally transduced and CCR1-CAR expression was assessed by flow cytometry. CCR1-CAR T cells promoted effective elimination of BM185 leukemia cells overexpressing murine CCR1 in vitro. Similarly, primary human T cells transduced with the CCR1-CAR exhibited potent cytotoxicity against C1498 leukemia cells overexpressing human CCR1 in vitro. To evaluate anti-leukemia activity of the CCR1-CAR T cells in vivo, BALB/c mice were sublethally irradiated and engrafted with 1x10 5 BM185 leukemia cells modified to overexpress murine CCR1. Two days later, mice were treated with 2x10 6 CCR1-CAR T cells and monitored for weight changes and survival. Growth of BM185 leukemia cells (expressing firefly luciferase) and expansion of CAR T cells (expressing gaussia luciferase) were assessed via two-color bioluminescence imaging (BLI). On day 6, mice were bled retro-orbitally and serum was analyzed for cytokine levels. The CCR1-CAR T cells demonstrated potent anti-leukemia activity and T cell expansion in vivo, promoting improved overall survival compared with control mice that received only tumor cells (Figure 1). CCR1-CAR T cell activity was accompanied by mild, self-limited toxicity, characterized by transient weight loss (Figure 2), but not significant elevation of serum levels of cytokines associated with cytokine release syndrome (CRS) such as IL-6 and TNF-α. BLI of the CCR1-CAR T cells revealed enhanced trafficking into the bone marrow and spleen followed by proliferation in irradiated mice even in the absence of tumor, suggesting that CCR1-CAR T cells are stimulated by CCR1 antigen expressed on non-leukemic host cells in addition to leukemia cells. CONCLUSIONS In summary, CCR1-CAR T cells represent a promising therapeutic strategy for AML that to our knowledge has not yet been evaluated in preclinical or clinical studies. We generated CCR1-CAR T cells that demonstrated potent anti-leukemia activity in vitro and in vivo in a syngeneic mouse model. Clinical signs of self-limited toxicity were not associated with elevated levels of inflammatory cytokines and might be caused by an on-target/off-tumor effect, which warrants further investigations to better characterize the safety profile of CCR1-CAR T cells. The CCR1-CAR T cells also displayed potent cytotoxicity in vitro via human CCR1, which will facilitate clinical translation of the findings. A CCR1-CAR may also offer benefit in other hematologic malignancies with reported CCR1 expression such as multiple myeloma or Hodgkin's lymphoma.

IMMU-04. LOW-INTENSITY PULSED ULTRASOUND ENHANCES CAR T CELL THERAPY BY INDUCING TWO-WAY IMMUNE CELL TRAFFICKING – T CELL INTO TUMOR AND MYELOID CELL INTO PERIPHERY IN A SYNGENEIC MOUSE GLIOBLASTOMA MODEL

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

Understanding the pathogenesis of brain arteriovenous malformation: genetic variations, epigenetics, signaling pathways, and immune inflammation.

Brain arteriovenous malformation (BAVM) is a rare but serious cerebrovascular disease whose pathogenesis has not been fully elucidated. Studies have found that epigenetic regulation, genetic variation and their signaling pathways, immune inflammation, may be the cause of BAVM the main reason. This review comprehensively analyzes the key pathways and inflammatory factors related to BAVMs, and explores their interplay with epigenetic regulation and genetics. Studies have found that epigenetic regulation such as DNA methylation, non-coding RNAs and m6A RNA modification can regulate endothelial cell proliferation, apoptosis, migration and damage repair of vascular malformations through different target gene pathways. Gene defects such as KRAS, ACVRL1 and EPHB4 lead to a disordered vascular environment, which may promote abnormal proliferation of blood vessels through ERK, NOTCH, mTOR, Wnt and other pathways. PDGF-B and PDGFR-β were responsible for the recruitment of vascular adventitial cells and smooth muscle cells in the extracellular matrix environment of blood vessels, and played an important role in the pathological process of BAVM. Recent single-cell sequencing data revealed the diversity of various cell types within BAVM, as well as the heterogeneous expression of vascular-associated antigens, while neutrophils, macrophages and cytokines such as IL-6, IL-1, TNF-α, and IL-17A in BAVM tissue were significantly increased. Currently, there are no specific drugs targeting BAVMs, and biomarkers for BAVM formation, bleeding, and recurrence are lacking clinically. Therefore, further studies on molecular biological mechanisms will help to gain insight into the pathogenesis of BAVM and develop potential therapeutic strategies.

Dual targeting ovarian cancer by Muc16 CAR T cells secreting a bispecific T cell engager antibody for an intracellular tumor antigen WT1.

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR T cell therapy in epithelial ovarian cancer and other cancers.

Efficacy and safety of glycosphingolipid SSEA-4 targeting CAR-T cells in an ovarian carcinoma model.

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized SSEA-4 cell-surface glycolipid as a target for CAR-T cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed pre-clinically in vitro and in vivo for anti-tumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4 positive cells in all the tested cancer cell lines whereas SSEA-4 negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific anti-tumor response at all the CAR-T cell doses used. At high T cell doses, CAR-T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR-T cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR-T therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

Chimeric Antigen Receptor T Cells in Glioblastoma-Current Concepts and Promising Future.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that is largely refractory to treatment and, therefore, invariably relapses. GBM patients have a median overall survival of 15 months and, given this devastating prognosis, there is a high need for therapy improvement. One of the therapeutic approaches currently tested in GBM is chimeric antigen receptor (CAR)-T cell therapy. CAR-T cells are genetically altered T cells that are redirected to eliminate tumor cells in a highly specific manner. There are several challenges to CAR-T cell therapy in solid tumors such as GBM, including restricted trafficking and penetration of tumor tissue, a highly immunosuppressive tumor microenvironment (TME), as well as heterogeneous antigen expression and antigen loss. In addition, CAR-T cells have limitations concerning safety, toxicity, and the manufacturing process. To date, CAR-T cells directed against several target antigens in GBM including interleukin-13 receptor alpha 2 (IL-13Rα2), epidermal growth factor receptor variant III (EGFRvIII), human epidermal growth factor receptor 2 (HER2), and ephrin type-A receptor 2 (EphA2) have been tested in preclinical and clinical studies. These studies demonstrated that CAR-T cell therapy is a feasible option in GBM with at least transient responses and acceptable adverse effects. Further improvements in CAR-T cells regarding their efficacy, flexibility, and safety could render them a promising therapy option in GBM.

Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR

The effectiveness of chimaeric antigen receptor (CAR) T cell therapies for solid tumours is hindered by difficulties in the selection of an effective target antigen, owing to the heterogeneous expression of tumour antigens and to target antigen expression in healthy tissues. Here we show that T cells with a CAR specific for fluorescein isothiocyanate (FITC) can be directed against solid tumours via the intratumoural administration of a FITC-conjugated lipid–poly(ethylene)-glycol amphiphile that inserts itself into cell membranes. In syngeneic and human tumour xenografts in mice, ‘amphiphile tagging’ of tumour cells drove tumour regression via the proliferation and accumulation of FITC-specific CAR T cells in the tumours. In syngeneic tumours, the therapy induced the infiltration of host T cells, elicited endogenous tumour-specific T cell priming and led to activity against distal untreated tumours and to protection against tumour rechallenge. Membrane-inserting ligands for specific CARs may facilitate the development of adoptive cell therapies that work independently of antigen expression and of tissue of origin.

Association of CAR-T cell secreting CD47 blocking protein with an enhanced antitumor activity in small cell lung cancer.

e20512 Background: Notch ligand family DLL3 has been considered a therapeutic target for small cell lung cancer. CD47/SIRPa is an important immunosuppressive factor in the tumor microenvironment and is considered to be a "checkpoint" of innate immune cells. Here, we described an DLL3 targeting CAR-T cells secreting CD47/SIRPa signal blocking protein. We hypothesized that the armored CAR-T cells, by blocking the CD47/SIRPa signal with specific antibodies may promote macrophages to engulf tumor cells and induce anti-tumor immune response, thus addressing a clinical need of cell therapy in SCLC. Methods: Bioinformatics analysis of immune cell composition in the microenvironment of small cell lung cancer, focusing on macrophage content and phenotypic characteristics. The CAR genes were cloned into the lentiviral vector pWPXLd, the expression, phenotype, activation, proliferation, killing and cytokine release of CAR-T cells were detected by flow cytometry, Western blot, RTCA and ELISA; NSG mice were injected subcutaneously with H446-DLL3 cells or H446-DLL3(+/-) combined with CAR-T cells or armored CAR-T cells. Tumor size was measured every 3rd day. The number and phenotype of macrophages in tumors were detected by FCM. Results: Bioinformatics analysis showed that tumors with high expression of DLL3 were often accompanied by downregulation of antigen presentation-related genes; there is a large amount of macrophage infiltration in SCLC, and the later the stage, the more the infiltration. CAR-T cells co-cultured with target cells could effectively activate T cells, promote CAR-T cell proliferation, secrete IL-2, IFN-γ and TNF-α cytokines, and specifically kill DLL3 positive tumor cells. We have established a solid tumor model with heterogeneous antigen expression. CAR-T cells can effectively inhibit the growth of tumors with positive DLL3 expression in vivo, but the inhibition on tumors with heterogeneous antigens is not obvious. Blocking CD47 can promote phagocytosis of tumor cells by macrophages. Western blot analysis revealed the presence of CD47 blocking protein CV1-Fc in the supernatant of secreted CAR-T cells, which can effectively promote the phagocytic effect of macrophages; Armored CAR-T cells have a better anti-tumor effect in antigen heterogeneous tumor model in vivo, delays tumor progression, and promotes the aggregation of M1 type macrophages. Conclusions: CAR-T cells that specifically target DLL3 can effectively kill antigen-positive tumor cells in vivo and in vitro. Blocking CD47 can promote the non-specific anti-tumor effect of macrophages. CAR-T cells secreting CD47 blocking protein have dual anti-tumor effects, not only killing antigen-positive tumor cells, but also recruit and activate macrophages to exert phagocytosis, and have better anti-tumor effects in antigenic heterogeneous solid tumors.

The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

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 1784: FGFR4 and CD276 dual targeting CAR T cells demonstrate synergistic antitumor activity in childhood rhabdomyosarcoma

Abstract Background Chimeric antigen receptor T cells (CAR T) are potential therapies for rhabdosarcoma (RMS), the most common soft tissue sarcoma in children, where patients with relapsed or refractory disease have a dismal cure rate, and effective therapies are urgently needed. RMS tumors express high levels of the cell surface receptors FGFR4 and CD276, and both are direct targets of the PAX3-FOXO1 chimeric oncogene. However, CAR T have shown poor performance in solid tumors due to T cell exhaustion, limited persistence, and heterogeneous expression of target antigens. We hypothesize that engineering CAR constructs targeting FGFR4 and CD276 will enhance their activity and dual targeting will overcome the heterogenous target expression to effectively eliminate RMS tumors. Methods We modified and tested different hinge and transmembrane domains (HTM) or intracellular domain (ICD) of FGFR4 CAR constructs and developed multiple dual targeting Bicistronic CARs (BiCisCARs) against FGFR4 and CD276. We tested these CARs in aggressive mouse models and performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of tumor infiltrating CAR T cells to functionally characterize these constructs. Results FGFR4 targeting CAR construct with CD8HTM and 4-1BB ICD could eliminate low burden RMS but was less effective against orthotopic tumors. Replacing CD8HTM with CD28HTM improved the efficacy in a moderate burden RMS model but did not show any benefit against a more aggressive RMS559 orthotopic model. Further modification using the CD28 ICD significantly enhanced the activity in a large burden RMS model but was unable to eradicate all tumors. Similarly, CAR T cells targeting CD276 alone, showed significant anti-tumor activity in moderate burden RMS models but could not eliminate all tumors. The BiCisCAR with FGFR4.CD28HTM.CD28ICD and CD276.CD8HTM.4-1BBICD was the most potent construct, eradicating 100% tumors in all tested orthotopic models, and those with heterogenous expression of target antigens. CITE-seq and flow cytometry assays demonstrated that this BiCisCAR showed the most significant tumor infiltration, and persistence, with limited exhaustion. This BiCisCAR exhibited a synergistic effect on cytokine production and anti-tumor activity, compared to single targeting CARs. Moreover, biochemical characterization revealed that the combined use of both CD28 and 4-1BB ICDs in the BiCisCAR resulted in the activation of three TCR downstream signaling pathways including AKT, Erk1/2 and p65. Conclusions and Future DirectionsThus, we have developed a potent BiCisCAR with dual targeting of FGFR4 and CD276 that shows optimal biochemical activity, persistence, and limited exhaustion, and addresses heterogenous expression of target antigens. This BiCisCAR will be further developed for future clinical trials in patients with high-risk RMS. Citation Format: Meijie Tian, Jun S. Wei, Adam Cheuk, David Milewski, Zhongmei Zhang, Yong Yuan Kim, Can Liu, Sherif Badr, Michael C. Kelly, Jerry T. Wu, Abdelrahman Rahmy, Hsien-Chao Chou, Xinyu Wen, Javed Khan. FGFR4 and CD276 dual targeting CAR T cells demonstrate synergistic antitumor activity in childhood rhabdomyosarcoma [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 1784.

Abstract 1149: Universal redirection of chimeric antigen receptor T cells against solid tumors via tumor cell membrane-inserting CAR ligands

Abstract Chimeric antigen receptor (CAR) T cell therapy is effective in treating certain hematological malignancies, but its translation to solid tumors has been challenging. Selecting an effective target antigen is hampered by heterogeneous tumor antigen expression and target antigen expression in healthy tissues. We describe a strategy to promote CAR T-mediated destruction of any tumor via intratumoral administration of a membrane-inserting fluorescein isothiocyanate lipid amphiphile molecule (“amph-FITC”) recognizable by a FITC-specific CAR. Firstly, we show labeling of tumor cells with amph-FITC in vitro, mediating cytotoxicity by FITC CAR T cells in coculture. Then, we demonstrate efficient tagging of tumor cells in vivo upon intratumoral injection with minimal labeling of non-tumor tissues, driving FITC CAR T cell proliferation and accumulation in tumors. Subsequently, we determine that this strategy stimulates an anti-tumor response that facilitates tumor regression in several syngeneic and human xenograft mouse models bearing different tissues of origin. In syngeneic models, this strategy induced the infiltration of host T cells and elicited endogenous tumor-specific T cell priming, leading to protection against tumor rechallenge. These results demonstrate the capacity to treat tumors independent of antigen expression or tissue of origin, addressing a major bottleneck in adoptive T cell therapy. Citation Format: Angela Zhang. Universal redirection of chimeric antigen receptor T cells against solid tumors via tumor cell membrane-inserting CAR ligands [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 1149.

Abstract 2013: Oxidized anthracycline payloads induce anti-tumor immunogenic cell-death and show linker-dependent tolerability when delivered as ADCs

Abstract Novel payloads with orthogonal mechanisms of action (MOAs) to established ADC technologies are important for expanding the utility of antibody-drug conjugates (ADCs) in cancer treatment. Anthracyclines, a class of cytotoxic small molecules that include important clinical chemotherapeutics such as doxorubicin, have long been of interest to the ADC field due to their high potency and unique MOA. Interest in anthracyclines has increased in recent years due to their status as canonical inducers of immunogenic-cell death (ICD), a feature that may lead to more durable tumor regressions and increased synergy with immune checkpoint blockade. In this work, we investigate several drug-linkers based on oxidized derivatives of the highly potent anthracycline analogue PNU-159682. Resultant ADCs are potent and immunologically specific with linker-dependent bystander activity in co-culture cytotoxicity assays displaying heterogeneous antigen (Ag+/Ag-) expression. Rodent in vivo anti-tumor activity and tolerability are shown with significant differences emerging between the toxicity profile of the cleavable and non-cleavable analogues. In vitro, tumor cells treated with anthracycline ADCs showed key hallmarks of ICD including markers of endoplasmic reticulum (ER) stress and the induction of ICD damage-associated molecular patterns (DAMPs) such as HMGB1 and ATP. In vivo mouse xenograft studies show increased tumoral F4/80+ macrophage infiltration by IHC in anthracycline ADC treated animals relative to controls. ADCs delivering monomethyl auristatin E (MMAE) show comparable activity in ICD assays, confirming MMAE as an ICD inducing ADC payload alongside canonical inducers such as anthracyclines. Our results indicate there is a therapeutic window in rodents alongside ICD induction by anthracycline ADCs, positioning anthracyclines as a potentially impactful class of ADC payloads. Citation Format: Joseph Z. Hamilton, Kerry Klussman, Rebecca Mahzereh, Jessica Simmons, Michelle Ulrich, Shyra J. Gardai, Peter D. Senter, Patrick J. Burke. Oxidized anthracycline payloads induce anti-tumor immunogenic cell-death and show linker-dependent tolerability when delivered as ADCs [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 2013.