Membrane-proximal Epitope Research Articles

Optimizing Siglec-8-Directed Immunotherapy for Eosinophilic and Mast Cell Disorders

Background/Objective: Current treatments for eosinophilic and mast cell disorders are often ineffective. One promising target to improve outcomes is sialic acid-binding immunoglobulin-like lectin-8 (Siglec-8). As limitations, there are few Siglec-8 monoclonal antibodies (mAbs) available to date, and Siglec-8-directed treatments have so far primarily focused on unconjugated mAbs, which may be inadequate, especially against mast cells. Methods: Here, we used transgenic mice to raise a diverse panel of fully human mAbs that either recognize the V-set domain, membrane-distal C2-set domain, or membrane-proximal C2-set domain of full-length Siglec-8 as a basis for novel therapeutics. Results: All mAbs were efficiently internalized into Siglec-8-expressing cells, suggesting their potential to deliver cytotoxic payloads. Tool T cell-engaging bispecific antibodies (BiAbs) and chimeric antigen receptor (CAR)-modified natural killer (NK) cells using single-chain variable fragments from Siglec-8 mAbs showed highly potent cytolytic activity against Siglec-8-positive cells even in cases of very low target antigen abundance, whereas they elicited no cytolytic activity against Siglec-8-negative target cells. Siglec-8V-set-directed T cell-engaging BiAbs and Siglec-8V-set-directed CAR-modified NK cells induced substantially greater cytotoxicity against cells expressing an artificial smaller Siglec-8 variant containing only the V-set domain than cells expressing full-length Siglec-8, consistent with the notion that targeting membrane-proximal epitopes enhances effector functions of Siglec-8 antibody-based therapeutics. Indeed, unconjugated Siglec-8C2-set mAbs, Siglec-8C2-set-directed T cell-engaging BiAbs, and Siglec-8C2-set-directed CAR-modified NK cells showed high antigen-specific cytolytic activity against Siglec-8-positive human cell lines and primary patient eosinophils. Conclusions: Together, these data demonstrate Siglec-8-directed immunotherapies can be highly potent, supporting their further development for eosinophilic and mast cell disorders.

The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.

Follow-up on phase 1 study of AT101, a novel anti-CD19 chimeric antigen receptor cell therapy (CAR-T) in patients with relapsed or refractory (r/r) b-cell non-Hodgkin lymphoma.

7035 Background: All the FDA-approved CD19 CAR-T products are based on FMC63 scFv, which binds to the membrane-distal region of CD19. We developed a novel anti-CD19 antibody clone (1218) binding to a membrane-proximal epitope of CD19 with fast on/off kinetics. AT101 is an autologous CAR-T cell transduced with a lentiviral vector encoding CAR comprised of a humanized scFv of 1218, 4-1BB costimulatory, and CD3zeta domain. Methods: In the phase 1 trial, patients (n=3 per dose level; up to n=18 in total) are treated with AT101 in 3 dose-escalation cohorts based on a standard 3 + 3 design. Each patient received a single intravenous dose of AT101 at dose level (DL) 1 (0.2 x 106 cells/kg), DL2 (1.0 x 106 cells/kg), or DL3 (5.0 x 106 cells/kg). The primary objective is to determine the safety, the maximum tolerated dose, and the recommended phase 2 dose of AT101. Key eligibility criteria include patients aged ≥19 with histologically confirmed relapsed or refractory B-cell non-Hodgkin lymphoma (NHL). Tumor responses were evaluated using Lugano 2014 criteria at 4 weeks before AT101 infusion as well as at 4 weeks and 3, 6, 9, 12, and 18 months after AT101 infusion. Results: From March 2022 to December 2022, fourteen patients were enrolled and 12 patients were treated, who were their median age of 62.5 years (range 39 to 84) and received a median of three prior lines of therapy (range 2-9). Their subtypes were as follows: diffuse large B cell lymphoma (DLBCL; n=7, 58.3%), follicular lymphoma (FL; n=3, 25.0%), mantle cell lymphoma (MCL; n=1, 8.3%), or marginal zone lymphoma (MZL; n=1, 8.3%)). The data collection cut-off date was January 31, 2024. Based on the best overall response up to three months, eleven patients responded with an overall response rate (ORR) of 91.7%, and a complete response (CR) was observed in nine patients (75%). Remarkably, in DL2 and DL3 groups, the CR was 100.0%. Among nine patients who achieved CR, seven patients have remained in CR during the median follow-up of 13.6 months (1.6-22.3 months). One patient experienced relapse and another one died from septic shock. The median duration of response (DOR), progression-free survival (PFS), overall survival (OS), and event-free survival (EFS) were 19.5, 17.2, 18.9 and 17.2 months, respectively. The median DOR, PFS, OS and EFS were not reached. Conclusions: In this first-in-human phase 1 trial, AT101 was tolerable with limited and manageable toxicities. In comparison to current FMC63 scFv-based CD19 CAR-T therapies, AT101 exhibited potent and more enduring efficacy with a remarkable suppression of relapse after CR. A phase 2 clinical trial is currently under-going for DLBCL patients. Clinical trial information: NCT05338931 .

Developing a membrane-proximal CD33-targeting CAR T cell

BackgroundCD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking.MethodsWe developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR...

Abstract 1884: DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies

Abstract CD56, also known as neural cell adhesion molecule 1 (NCAM1), is a type I plasma membrane glycoprotein involved in cell-cell and cell-matrix adhesion. It is overexpressed in almost all neuroblastomas, 95% of small cell lung cancers and 78% of multiple myeloma patients[1], whereas CD56 is less frequently or less abundantly expressed in normal tissues, making it a potential target for tumor therapy. So far, no CD56-targeting ADCs have entered late-stage clinical trials.DXC006 is an antibody-drug conjugate of a humanized anti-CD56 antibody (DXA006) linked to the topoisomerase I inhibitor CPT113 through a cleavable peptidyl linker and CROSSCONJU™ technology. The pharmacologic activity and mechanism of action of DXC006 were investigated in several human cancer cell lines and xenograft mouse models. Pharmacokinetic and safety profiles were also assessed in rats and cynomolgus monkeys.DXC006 binds specifically to CD56 and internalizes into tumor cells, after intracellular trafficking to lysosome and CPT113 releasing, induces DNA damage. In vitro cell based assays showed that DXC006 remarkably inhibited the cell growth of CD56-positive cell lines of IMR-32 (neuroblastoma), NCI-H526 (small cell lung cancer), and NCI-H929 (multiple myeloma), with IC50 of 0.0046~0.061 nmol/L. Although DXC006 also binds to CD56 expressed NK cells, we found that the inhibition of DXC006 to human NK cells was much less effective, with IC50 higher than 675 nmol/L, which is ten thousand-folds less potent than that of targeted abnormal cells. In vivo, DXC006 demonstrated good durable antitumor activities in three CD56-positive xenograft models of IMR-32, NCI-H526 and NCI-H929 cell lines, with a single dose of 2 mg/Kg, 3 mg/Kg, and 5 mg/Kg, respectively. No animal death or moribund outcome was observed in two months during preclinical safety studies in 150 mg/Kg Q2W dosed rats. For cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of DXC006 was 20 mg/Kg, in Q2W, four times of administration. PK profiles of both DXC006 the naked antibody DXA006 were comparable in both rats and monkeys. In summary, DXC006 demonstrated strong antitumor activity in various types of cancer cells both in vitro and in vivo, and good pharmacokinetic and safety profiles in rat and monkeys, a potential to be explored in clinical applications. [1] Yang Feng, Yanping Wang, Zhongyu Zhu, et al. Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal nonoverlapping epitopes. mAbs 2016; 799-810. Citation Format: Xiangfei Kong, Huihui Guo, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Fang Du, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies [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 1884.

Abstract 47: Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy

Abstract Introduction: Genetically engineered T cells expressing chimeric antigen receptors (CARs) have shown substantial anti-tumor efficacy in hematological malignancies during recent years. As the field evolves to novel target epitopes, longer CAR structures are needed to reach membrane proximal epitopes for improved efficacy. Our approach to reach such epitopes is to have (non-functional) SIRPalpha based extracellular extension, spacer, to our CAR structure. We have shown, in this proof-of-concept study, that stability of the extracellular spacer of a CAR plays a pivotal role to improve in vivo anti-tumor efficacy. Various SIRPalpha-based CARs, FiCARs, targeting CD19 was studied for their function. Protein stability of the CD19 CAR structures were characterized and subsequently the killing efficacy of target cells and cytokine release in in vitro as well as in vivo anti-tumor efficacy was evaluated. Methods & Results: In vitro co-culture assays with primary CD19 CAR T and CD19 positive cancer cells had shown no significant differences between the different spacer containing FiCAR structures. A dose dependent killing efficacy was observed in all the tested structures with up to >90% killing of NALM6 and Raji cells. Comparable results for IL-2, IFN-gamma, TNF-alpha, IL-4, IL-13 and granzyme B response were seen in co-cultured assay. In the protein stability studies, modifications in the extracellular part of the CAR structure led to an increase in thermal stability (over 2 °C) and reduced/halted multimer formation. To study the FiCAR T cell function in an in vivo tumor model, NSG mice were injected i.v with Raji cells and few days later mice were injected i.v with FiCAR T cells. Anti-tumor efficacy and overall survival were studied at the end of the study (60 days), the enhanced stability of FiCAR T structure translated into improved anti-tumor efficacy and survival. Interestingly, none of the mice survived in the least stable structure, 40% survival with intermediate stability structures and over 70% survival was observed in most stable structures. Conclusion: A stabilized SIRPalpha-based CD19 FiCARs dramatically enhance anti-tumor efficacy and survival in Raji tumor model. The stabilization is critical for improved efficacy, especially in the in vivo setting. This data suggests that, in addition to the length of the CARs stability of the CAR structure appear to play a crucial role in the efficacy of the CAR T cells. The modifications presented here enhanced CD19-targeting CAR T cell efficacy. This approach requires further investigation to improve the efficacy of currently approved CAR T cells, especially CD19-CAR T cells. Citation Format: Henrik Paavilainen, Jan Koski, Veera Nikoskelainen, Manar Elmadani, Endrit Elbasani, Can Hekim, Emmi-Leena Ihantola, Virginie Kergourlay, Pirjo Vehmaan-kreula, Marie Nyman, Kiira Kalke, Markus Nurmi, Hector Monzo, Paivi Ojala, Anu Autio, Matti Korhonen, Anil Thotakura. Stabilization of long CAR T cells is crucial for in vivo antitumor efficacy [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 47.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

Chimeric antigen receptor (CAR-) T cell therapies targeting B-cell restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B-cell malignancies, but relapse remains common. Camelid single domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28 to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, though only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies a lead CD22sdCAR-T was selected; achieving comparable complete responses to a benchmark short-linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular level specificity of the lead CD22-sdAb. This presents a complete report on pre-clinical development of a novel CD22sdCAR therapeutic.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

BackgroundCommercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy.MethodsWe developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL.ResultsPreclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia.ConclusionsWe developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL.Trial registrationNCT05338931; Date: 2022–04-01.

Development of a Regulated, Optimized Mesothelin Chimeric Antigen Receptor (CAR) for the Treatment of Mesothelin Positive Cancers

Introduction: Mesothelin (MSLN) is an attractive target for chimeric antigen receptor T cell (CAR-T) immunotherapy as it is highly expressed on mesothelioma, ovarian, lung cancer, and some forms of leukemia, including approximately 30% of acute myeloid leukemia (AML); however, MSLN directed CAR-T cells are prone to exhaustion, and soluble proteins, such as shed MSLN and MUC16 can inhibit anti-tumor efficacy. To address these deficiencies, we developed a novel, fully-human MSLN-specific CAR using our OutSpacer™ technology to optimize the immune synapse geometry. Further, this MSLN CAR is expressed using a novel regulatory response element to dynamically express the CAR molecule to enhance CAR-T potency and durability. Methods: A set of fully-human single domain binders (VHH) were produced and screened against a spacer library to identify high-performing CARs. The spacer library is composed of human extracellular polypeptides selected for optimal biophysical properties (e.g., flexibility), enabling systematic sampling to identify CAR constructs with optimal immune synapse geometry. More than 300 MSLN-directed CAR candidates were evaluated in vitro using 2D or 3D (spheroid) culture assays to measure tumor killing and cytokine production against several MSLN + cell lines that express different levels of the target protein. These functional assays were also performed in the presence of recombinant MSLN (10ug/mL) or MUC16 (1ug/mL) to assess potential inhibition by shed target or ligand. Top performing MSLN CARs were subsequently tested in NSG mice (2e6, 4e6 and 8e6 cells/animal; 2 donors) subcutaneously engrafted with MSLN + H1650 tumor cells to identify the best CAR candidate. To further improve the durability of T cell response, the MSLN CAR was expressed under the control of an engineered promoter (OP1) to dynamically regulate expression, and compared to CARs expressed constitutively using the MND promoter. CAR-T cells were repetitively stimulated (4x) by plate-bound antigen in combination with CD58 and ICAM1, and subsequently tested in a tumor spheroid assay for killing, cytokine production, CAR expression and T cell activation/ exhaustion markers (i.e., PD-1, TIGIT, CD39 and TIM3). Results: Anti-MSLN VHH domains targeting region 1 or 3 (RI and RIII) of the MLSN extracellular domain were paired with a defined spacer library to define the optimal immune synapse. Contrary to the conclusions of prior studies, RI (membrane-distal) binders outperformed RIII (membrane-proximal) binders when paired with their optimal spacer. Based on tumor killing and cytokine production, RI targeted CARs preferred a spacer length of approximately 75 Å, consistent with our previously-reported optimal CAR synapse of ~200 Å. All spacer optimized MSLN-specific CARs resisted inhibition by soluble MSLN and MUC16. In vivo studies with two donors in NSG mice engrafted with MSLN + tumors showed that one CAR (B8S8) eliminated tumors at a dose of 2e6 and 4e6 CAR-T cells per animal, where other CAR constructs, including a clinically-used MSLN-specific CAR, failed to control tumors even at 8e6 cells per animal. Secondly, we assessed whether a novel “stim-on” promoter (OP1) could enhance CAR-T activity by preserving T cell function. Here, B8S8 CAR was expressed via OP1 or the constitutive MND promoter (n=3 donors). Following chronic antigen exposure by plate-bound antigen, OP1 regulated MSLN CAR T cells retained the capacity to eliminate MSLN + H226 tumor spheroids compared to MND-driven CAR T constructs. OP1-regulated CAR T cells also continued to produce high levels of cytokines (IFN-γ and IL-2) whereas MND CAR-T cells lost cytokine production ability. Further, phenotypic analyses showed that MND CAR-T cells expressed elevated levels of PD-1 and TIGIT compared to OP1 CAR-T cells. Conclusions: Contrary to the conclusions of prior studies with MSLN-targeted CARs, membrane distal epitopes were superior to membrane proximal epitopes when binders were paired with the correct spacer, and shed MSLN and MUC16 were not inhibitory for the best-performing CARs, regardless of target epitope. Furthermore, regulated expression enhanced the potency and durability of response compared to constitutive expression. This approach may improve CAR activity in patients with MSLN + solid tumors or leukemia.

A First-in-Human Phase I Study of AT101, a Novel Anti-CD19 Chimeric Antigen Receptor T Cell Product Targeting a Membrane-Proximal Domain of CD19 in Adults with Relapsed or Refractory B Cell Non-Hodgkin Lymphoma

Introduction: Anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are highly efficacious against advanced B cell non-Hodgkin lymphoma (NHL) but the majority of patients (pts) ultimately fail. Several mechanisms contribute to CART19 failure, including CD19-neg escape and CART dysfunction. Notably, all four commercial CART19 products utilize the FMC63 single chain variable fragment (scFv) with high-avidity specificity to a CD19 membrane-distal epitope. Interestingly, loss of the FMC63-recognized epitope due to CD19 mutations ( Zhang Z, JITC, 2020) or epitope-masking (CAR19:CD19) ( Ruella M, Nat Med, 2018) has been described. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 with low avidity could: 1. mitigate CD19 epitope loss; but also 2. enhance CART functions ( Fig. 1A) . Methods and results: We developed an autologous, CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218) specific to a membrane-proximal epitope of CD19 (amino acids T51,S53, E55,K59 and K63). We previously reported initial preclinical data ( Patel R., ASH, 2021 #2798) that demonstrated h1218-CART19 recognize and kill B-cell leukemia that aberrantly expresses the FMC63-CAR19. More recently, we demonstrated that differently than FMC63-based CART, h1218-CART19 could respond to and kill tumor cells expressing point mutations of CD19 (L174V and R163L). Of note, h1218-CART19 had enhanced control of CD19+ B-cell neoplasms as compared to FMC63-CART19 both in vitro and in vivo. Mechanistically, thanks to the lower avidity and faster on/off rate, h1218-CART19 had decreased T cell exhaustion and activation-induced cell death. Given the promising preclinical results, we tested the clinical h1218-CART19 product (AT101) in a first-in-human, multi-center, phase I/II, dose-escalation clinical trial for pts with histologically confirmed relapsed or refractory B cell NHL (NCT05338931). For the phase I study, pts were treated with a single intravenous infusion of AT101 (day 0) in three dose levels (DL) based on a standard 3+3 design: DL1 (0.2 x 10 6 cells/kg), DL2 (1.0 x 10 6 cells/kg), or DL3 (5.0 x 10 6 cells/kg). All pts received lymphodepletion with intravenous fludarabine (250 mg/m 2) and cyclophosphamide (25 mg/m 2) on days -4, -3, and -2. The primary objective was to determine the maximum tolerated dose (MTD) of AT101. The secondary objectives were to evaluate AT101 pharmacokinetics and preliminary efficacy. At the July 3, 2023 data cut, 12 pts (median age 62.5 years (range 39-84); diffuse large B cell lymphoma (DLBCL, n=7, 58.3%), follicular lymphoma (FL, n=3, 25.0%), mantle cell lymphoma (MCL, n=1, 8.3%), and marginal zone lymphoma (MZL, n=1, 8.3%)) received AT101 ( Fig. 1B). The median follow-up after AT101 was 6.5 months (1.5-13.7 months), the median number of previous lines of treatment 3 (range 1-8); no prior cellular therapies. Five pts (41.7%) had previous autologous stem cell transplantation. Two pts (16.7%) had refractory disease and two pts (16.7%) received bridging therapy. Three pts (2 pts at DL2 and 1 pt at DL3) developed a grade (G) 1 cytokine-release syndrome (CRS) and one pt at DL3 developed a G3 CRS that resolved within 24 hours. Three pts (1 pt at each DL) developed immune-cell-related neurotoxicity syndrome (ICANS), of which one G4 dose-limiting toxicity (DLT) was reported at DL1 that corrected within 6 days. One DL3 pt experienced G3 sepsis that promptly resolved; the same pt ultimately developed fatal neutropenic septic shock outside the DLT period. Four additional patients experienced ≥G3 neutropenia without evidence of infection. Complete response (CR) occurred in 8/12 pts (66.6%) and overall response (CR+partial response) in 83.3% (95%CI, 51.6-97.9) at day 28 post-AT101 infusion ( Fig. 1B). Remarkably, in DL2 and DL3, the CR rate was 100.0%. Of the 8 pts in CR none has relapsed (median follow-up 6.0 months). AT101 cells expanded (peak in DL3 113.6 ± 6.50 x10 4 CAR gene copies/ug DNA) and persisted in patients' blood. Conclusion: In this first-in-human phase I trial, AT101 was tolerable with limited and manageable toxicities. Notable preliminary efficacy was observed at the RP2D with no evidence of disease relapse after achieving a CR. These results warrant the pursuit of the planned phase II expansion cohort.

Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P<.001, 947 P<.003, 977 P<.0002, 980 P<.008 BCMA P<.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

Novel mesothelin antibodies enable crystallography of the intact mesothelin ectodomain and engineering of potent, T cell-engaging bispecific therapeutics

Mesothelin is a glypiated, cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed by many cancers. Implicated in cell adhesion and multiple signaling pathways, mesothelin’s precise biological function and overall structure remain undefined. Antibodies targeting mesothelin have been engineered into immunotoxins, antibody-drug conjugates, CAR-T cells, or bispecific T cell engagers as candidate therapeutics but most face challenges, including binding epitopes that are not optimal for selected modalities. Here we describe the isolation and characterization of a novel anti-mesothelin antibody, 1A12, including crystallographic mapping of the 1A12 epitope in relation to other antibodies (amatuximab, anetumab). 1A12 possesses uniquely favorable properties, including a membrane-proximal epitope, and enabled structure determination of the complete mesothelin ectodomain. We incorporated 1A12 into two different bispecific T cell engaging architectures with various anti-CD3 co-targeting elements as candidate therapeutics, demonstrating in vitro functionality and potency.

Efficient chimeric antigen receptor targeting of a central epitope of CD22

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and B-cell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to targetalternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane-proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971-CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patient-derived xenograft samples. Side-by-side comparisons indicated that while IS7-CAR killed less rapidly than m971-CAR invitro, it remains efficient in controlling lymphoma xenograft models invivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

A phase 1 study of AT101, a novel anti-CD19 CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma.

7522 Background: All the FDA-approved CD19 CAR-T products are based on FMC63 scFv, which binds to the membrane-distal region of CD19. We developed a novel anti-CD19 antibody clone (1218) that binds to a membrane-proximal epitope of CD19, thereby not competing with FMC63. AT101 is an autologous CAR T cell transduced with a lentiviral vector, including the CD19-CAR with a humanized scFv of 1218, 4-1BB costimulatory, and CD3zeta domain. Methods: In this phase 1 trial, patients (n = 3 per dose level; up to n = 18 in total) are treated with AT101 in 3 dose-escalation cohorts based on a standard 3 + 3 design. Each patient received a single intravenous dose of AT101 at dose level (DL) 1 (0.2 x 106 cells/kg), DL2 (1.0 x 106 cells/kg), or DL3 (5.0 x 106 cells/kg). The primary objective is to determine the safety, the maximum tolerated dose (MTD), and the recommended phase 2 dose (RP2D) of AT101. The secondary objective is to evaluate the pharmacokinetics of AT101 and the preliminary efficacy, such as overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), event-free survival (EFS), and overall survival (OS). Key eligibility criteria include patients aged ≥19 with histologically confirmed relapsed or refractory B-cell non-Hodgkin lymphoma (NHL). Results: Fourteen patients were enrolled from March 2022 to December 2022, and nine were treated. The median age of treated patients was 61.6 years (ranged from 39 to 84) after a median of 4 prior lines of therapy (range 2-10). Their subtypes of NHL were as follows: diffuse large B cell lymphoma (n = 4), follicular lymphoma (n = 3), mantle cell lymphoma (n = 1), or marginal zone lymphoma (n = 1). The dosing of AT101 at DL1 and DL2 was completed. The dosing at DL3 is ongoing. Across cohorts 1 and 2, no grade 3 or higher cytokine release syndrome (CRS) was reported. Among the first three patients at DL1, one dose-limiting toxicity (DLT) of grade 4 neurotoxicity was observed but resolved in a week without sequelae. No other DLTs were observed in the additional three patients at the DL1 and three at the DL2 cohort. Another one at DL2 experienced grade 1 CRS with grade 1 neurotoxicity. Five patients experienced Grade ≥3 hematologic toxicities. An ORR is 66.7% (4/6) in cohort 1 and 100% (3/3) in cohort 2, including six complete responses (CR, 50.0% [3/6] in cohort 1 and 100% [3/3] in cohort 2). As of February 13, 2023, all six CRs are ongoing, including two patents exceeding six months after the treatments. Updated results will be presented at the meeting, including the cohort of DL3. Conclusions: In this phase I study, AT101 was well tolerated at the first two dose levels and showed promising efficacy in relapsed or refractory B-cell NHL patients. The majority of adverse effects were transient and manageable. The administrations of DL3 are currently ongoing, and updated results will be presented at the meeting. Clinical trial information: NCT05338931 .

Hinging on Success: Leveraging the Power of CAR T-Cell Therapy through In-Silico Modeling of Hinge Length and Epitope Location

Abstract Genetic engineering of T-cells using chimeric antigen receptors (CARs) has shown promise in the treatment of relapsed/refractory B-cell leukemia. However, a significant percentage of patients will eventually relapse, often due to the emergence of tumor cells expressing low levels of the target antigen. Previous research has demonstrated that the hinge domain of the CAR plays a crucial role in regulating the cytotoxic capabilities of these cells. In this study, we assessed the potential to model optimal hinge length for a given single-chain variable fragment (scFv) in relation to the location of the targeted epitope. We designed an array of hinges originating from CD8α and incorporated these hinges into CARs targeting CD19, CD22, CD33, and CD20 antigens. We found that the location of the epitope is critical in determining the optimal hinge; a narrow range of short hinges is more effective against membrane-distal epitopes while long hinges are more effective against membrane-proximal epitopes. To build an in-silico model, we used AlphaFold2 (AF2), reducing the length of the multiple sequence alignment (MSA) clusters to introduce stochasticity and increase the conformational diversity of the structural predictions for the CAR hinges and target proteins. We discovered that CARs with a predicted intermembrane distance of 15nm between the CAR and ligand were particularly effective against antigen-low leukemic cells. Our findings suggest that intermembrane distance can predict CAR responsiveness against a specific epitope, enabling the rapid optimization of CARs by adjusting hinge length. The development of this modeling strategy will facilitate the design of CARs with optimized cytotoxic potential against a wide range of novel targets. Intramural Research Program of the National Cancer Institute (Bethesda, MD)

Abstract CT130: An open label, dose escalation, phase 1 study of AT101, a novel CD19-directed CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma

Abstract Background and Preliminary Data: All the FDA-approved CD19 CAR-T cell therapies are based on an antigen-binding domain (scFv) based on the FMC63 antibody which binds to the membrane-distal region of CD19 to an epitope encoded by exons 3 and 4 (Klesmith JR, Biochemistry, 2019; Zhang Z, JITC, 2020). While these CART19 products are very effective in the clinic, the majority of patients still do not respond or eventually relapse due to several mechanisms of resistance, including T cell dysfunction and epitope CD19-negative escape. Novel strategies to enhance the activity of CART cells and reduce escape are critically needed. We recently demonstrated that modifications of the binding region of the CAR (scFv) (Singh N., Nat Med, 2021) can drastically change the interaction between the CAR T cell and the cancer cells, potentially improving the anti-tumor effect. To this goal, we developed a novel anti-CD19 antibody clone (1218) that binds to a membrane-proximal epitope of CD19 (exon 2 region K59-K63) thereby not competing with FMC63. We developed a novel CART19, called AT101, using a humanized 1218 scFv along with 4-1BB costimulatory and CD3zeta domain in a lentiviral backbone. In preclinical models, AT101 showed more potent in vitro cytotoxicity against CD19-positive B lymphoma cells in a long-term killing assay and in a B-ALL (NALM6) in vivo model as compared to the control of FMC63 based CAR-T cells. In addition, differently than FMC63-based CART, AT101 could target tumor cells expressing point mutations of CD19 that are associated with relapse post-CART19 (FMC63) (Zhang Z, JITC, 2020) and leukemic blasts aberrantly expressing FMC63 CAR19 on their surface (Ruella M, Nat Med, 2018). Based on the preclinical efficacy and safety, a phase 1 clinical trial testing autologous AT101 was started for patients with relapsed and refractory B-cell non-Hodgkin lymphoma. Trial Design and Methods: This open-label, multi-center, first-in-human Phase 1 study will assess the safety and feasibility of AT101 in patients with relapsed or refractory B cell non-Hodgkin lymphoma. Key eligibility criteria include patients aged ≥19 years of age with histologically confirmed relapsed or refractory aggressive B-cell non-Hodgkin lymphoma. In this phase 1 trial, patients (n=3 per dose level; up to n=18 in total) are treated with AT101 in 3 dose-escalation cohorts based on a standard 3 + 3 design. CART doses are 2.0 × 105, 1.0 × 106, or 5.0 × 106 CAR+T cells/kg. The primary objective is to determine the safety, the maximum tolerated dose (MTD), and the recommended phase 2 dose (RP2D) of AT101 in participants following lymphodepletion with cyclophosphamide and fludarabine (250 mg/m2 and 25 mg/m2). The secondary objective is to evaluate the preliminary efficacy assessments (overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), event-free survival (EFS), and pharmacokinetics of AT101. Exploratory objectives include assessment of CD19 expression and cytokines in the blood. Patients will be followed for safety for at least 60 months post AT101 infusion. Clinical trial registry number: NCT05338931. As of January 11, 2023, AT101 has been infused to six patients in cohort 1 and three patients in cohort 2. Detailed results will be presented at the meeting. Citation Format: Yunlin Zhang, Ki Hyun Kim, Dok Hyun Yoon, Ruchi P. Patel, Jae-Cheol Jo, Hyungwoo Cho, Jong-Ho Lee, Hyun-Jong Lee, Lei-Guang Cui, In-Sik Hwang, Young Ha Lee, Jong-Hoon Kim, Yong Gu Lee, Puneeth Guruprasad, Jong-Seo Lee, Junho Chung, Marco Ruella. An open label, dose escalation, phase 1 study of AT101, a novel CD19-directed CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma [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 CT130.

An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers

T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of Tcell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. Invitro, our lead compound CC-3 showed superior tumor cell killing, Tcell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. Invivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced β catenin levels in HCC cells. Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

Pharmacological Modulation of CD21 Antigen Density Enhances Chimeric Antigen Receptor (CAR-T) Cell Function

Introduction Antigen density has emerged as an important factor regulating CAR-T cell activity with low antigen density being associated with reduced potency in pre-clinical models across several targets. In clinical trials of CD22 CAR-T cells, the low and heterogeneous CD22 expression in B-ALL enabled CD22lo blasts to escape CAR-T cell detection leading to antigen dim relapses (Nat Med. 2018, 24:20-28). We have previously reported on the development of novel CD21 directed CAR-T cells (CAR21) for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) (Blood. 2021, 138:902). Donor derived CAR21 T cells targeting membrane proximal epitopes and utilizing a Fab-CAR architecture were capable of specific cytotoxicity and cytokine secretion against CD21 positive target cells in vitro and were active in vivo. However, in co-culture assays, CAR21 cells secreted less IL-2 against cell lines and primary tumors expressing CD21 at very low antigen density (<1000 copies/cell). We have shown that CD21 is expressed in ~60% of primary T-ALL samples (n=58) although at low antigen density (mean 1262 copies/cell) compared to more established targets such as CD19. We wanted to explore the impact of this low antigen density on CD21 CAR-T function and whether it was possible to upregulate CD21 density by pharmacological means. Results We have previously shown that CD21 is a direct transcriptional target of the NOTCH-MYC pathway and that PI3K/mTOR inhibition of T-ALL results in activation of this pathway and consequent CD21 up-regulation (Leukaemia. 2013, 27:650). We therefore incubated six T-ALL cell lines with four inhibitors of the PI3K/mTOR/AKT pathway (AZD5363, GDC0941, BEZ235 and rapamycin) for 48 and 72hrs and showed surface CD21 up-regulation by flow cytometry of > two-fold at both time points for all inhibitors, with BEZ235 showing the greatest fold increases across all cell lines. Incubation with the gamma-secretase inhibitor L685 resulted in significantly down regulated CD21 expression across all cell lines in keeping with the known NOTCH regulation of CD21 expression. We were next interested in the persistence of this enhanced CD21 expression after drug withdrawal. In two T-ALL cell lines, PF382 and RPMI8402, the impact of BEZ235, rapamycin and L685 was sustained at 48hrs after washing off. PI3K/ mTOR mediated CD21 manipulation did not occur in a CD21 negative T-ALL cell line, normal donor B and T cells or B-ALL patient samples suggesting the effect was specific to T-ALL. When clinically approved PI3K inhibitors (Idelalisib, Duvelisib, Copanlisib, Everolimus) were used, an increase in CD21 expression was again seen across 5 T-ALL cell lines tested. Greatest fold increases (> 2-fold) in CD21 expression were seen with the dual PI3Kα and PI3Kδ inhibitor copanlisib and the mTOR inhibitor rapamycin. No consistent up regulation of CD7 or CCR9 by flow was seen after 48-hour copanlisib incubation suggesting that the effect was specific to CD21. PI3K inhibition was additionally able to mediate 2-fold increases in CD21 expression in a CD21 low patient-derived xenograft T-ALL sample. Further, in a MOLT-4 murine xenograft model of T-ALL, 2 doses of copanlisib were able to produce a 2-fold increase in CD21 expression. These PI3K-mediated increases in CD21 antigen density translated into improved in vitro CAR21 functionality. Across 3 T-ALL cell lines - MOLT-4, CUTLL-1 and RPMI-8402, 48hr pre-incubation with both rapamycin and copanlisib prior to co-culture with CAR21 or CAR19 cells, resulted in improved CD21 specific cytotoxicity, IFN-G and IL-2 release. However, in the same co-culture assay, when copanlisib was added directly to the co-culture rather than via pre-incubation, there was a significant attenuation of IFN-G and IL-2 production by CAR21 suggesting a direct inhibitory effect on CAR-T effector functions in keeping with the known immunosuppressive effects of PI3K inhibitors. Conclusion In summary, we have shown that CD21 expression in T-ALL can be increased through PI3K pathway inhibition and that enhanced CD21 antigen density through pharmacological priming results in improved in vitro CAR21 efficacy. In vivo models analyzing the impact of copanlisib tumor priming on CAR-T efficacy and persistence are ongoing. Target antigen modulation is a promising strategy for enhancing CAR efficacy and reducing risk of antigen negative relapse in low antigen density targets.