The Enhanced Functionality of Low-Affinity CD19 CAR T Cells Is Associated with Activation Priming and Polyfunctional Cytokine Phenotype

Abstract

We have recently described a low-affinity second-generation anti-CD19 Chimeric Antigen Receptor (CAR) (CAT), characterized by faster antigen dissociation rate which showed enhanced expansion, cytotoxicity and anti-tumour efficacy compared with the high affinity (FMC63 based) CAR used in Tisagenlecleucel in pre-clinical models. Furthermore, CAT CAR T cells showed an excellent toxicity profile, enhanced in vivo expansion and long-term persistence in a Phase I clinical study (Ghorashian et al Nature Med 2019). However the molecular mechanisms behind the improved properties of CAT CAR T cells remain unknown. Herein, we performed a systematic in vitro characterization of the transcriptomic (bulk RNA-seq) and protein (CyTOF) changes occurring in CAR T cells expressing a low-affinity (CAT) vs high affinity (FMC63) anti-CD19 CARs following stimulation with CD19 expressing targets. Untransduced (UT) controls and T cells lentivirally transduced to express CAT or FMC63 CD19 CARs were compared both at baseline and following stimulation with CD19+ Acute Lymphoblastic Leukaemia cell line NALM6. In Principal Component Analysis for both RNA-seq and protein results, we found that the major variance across conditions was explained by CD19-mediated CAR T activation. Strikingly, unstimulated CAT CAR T cells showed an intermediate degree of activation between UT T cells and antigen stimulated CAR T cells. Indeed, when comparing RNA-seq results of unstimulated CAT vs FMC63, we found enhanced expression (FDR <0.1) of genes involved in cytotoxicity (GNLY, GZMK) and T cell activation (HLA-DRA and HLA-DPA1) (Figure 1a), confirmed at protein level by CyTOF. This "activation priming" observed in CAT CAR T cells was associated with and may be driven by residual CD19-expressing B-cells present in the manufacture product, preferentially inducing a T Central Memory (TCM) phenotype in CAT vs FMC63, in both CD4 and CD8 T cells. Such priming is likely to be instrumental to CAT CAR T cells more potent cytotoxic response upon NALM6 stimulation, when they displayed further increase in the expression of immune stimulatory cytokines (IFNG, CSF2), chemokines (CCL3L1, CCL4, CXCL8) and IFNg responsive genes (CIITA) by RNA-seq, as well as augmented T cell activation (CD25, NFAT1) and proliferation (pRB) markers by CyTOF. To identify the mechanisms underlying the stronger basal activation of CAT CAR T cells, we analysed cytokine expression at the single cell level by mass cytometry. Interestingly, rather than an increment in the expression of individual cytokines, we found that the distinctive feature of CAT CAR T cells was a shift toward a cytokine polyfunctional phenotype, with a marked increase in the proportion of cells co-expressing 3 or more cytokines (17.50% CAT vs 7.33% FMC63) (Figure 1b). Of note, cytokine polyfunctionality (expression of more than 1 cytokine/cell) in pre-infusion CAR T cell products has been associated to improved clinical efficacy. The functional phenotype observed in CAT CAR T cells was linked to the preferential activation of the p38 MAPK phospo-signalling, which is activated downstream of TCR CD3ζ chain (present in the CARs) but is also central to cytokine-dependent T cell activation in memory T cells. Interestingly, cytokine polyfunctional CAT CAR T cells were enriched in the CD3+CD19+ trogocytic (trog+) population, found at higher proportion in CAT vs FMC63 at 24h post antigen stimulation. Although trogocytosis has been associated to CAR T cell fratricide killing, trog+ CAT CAR T cells displayed higher levels of proliferation (pRB), activation (CD25, NFAT1) and cytotoxic (Granzyme B, Perforin B) markers, pointing at a stimulatory role of trogocytosis over fratricide killing, potentially due to the low-affinity CAR T cells distinctive property of better discriminating between low (trog+ CAR T cells) and high (tumour cells) target expression levels. In conclusion, we described the molecular mechanisms underlying the low affinity CAT CAR T cells functional phenotype. Our results show that the potent and long-term anti-tumour responses observed with CAT may be sustained by the establishment of CAR T cells self-reinforcing circuits activated through polyfunctional cytokine crosstalk. This work may inform the future design of versatile CAR T cells, capable of balancing safety, efficacy and long-term persistence. Disclosures Ghorashian: Amgen: Honoraria; UCLB: Patents & Royalties; Novartis: Honoraria. Pule:Autolus: Current Employment, Other: owns stock in and receives royalties, Patents & Royalties; UCLB: Patents & Royalties; Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL.

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity

“…Many efforts are ongoing to enhance CART cell in vivo expansion and anti-tumor activity by using strategies to non-specifically stimulate CART cell proliferation or re-stimulate CART cells after infusion, utilizing synthetic biology or combination therapy to edit their exhaustion pathways or prevent their apoptosis. 32 , 33 However, this often improves CART efficacy at the cost of the CART safety profile: most of these approaches are associated with increased production of inflammatory cytokines and higher risk for the development of CART-associated toxicities such as CRS and NT (efficacy/toxicity linkage). In contrast, our data indicate that GM-CSF knockout in CART cells results in enhanced CART cell proliferation and anti-tumor activity, while being associated with a marked reduction in GM-CSF levels.…”

“… 32 In a different approach, modification of CART cell activation has been accomplished through modulation of CART cell antigen binding by using a lower affinity single chain variable fragment in the CAR design. 33 However, these strategies – aimed at ameliorating CART cell apoptosis and nonspecifically enhancing their proliferation – are associated with increased inflammatory cytokine secretion and CART cell-associated toxicities.…”

Tyrosine kinase inhibitor maintenance following chimeric antigen receptor T‐cell therapy in Philadelphia chromosome‐positive acute lymphoblastic leukaemia

“…To the editor, Chimeric antigen receptor (CAR) T-cell therapy produces high response rates in acute lymphoblastic leukaemia (ALL), and while products with 4-1BB costimulatory domains or those with low affinity are persistent, other products have limited response durability without subsequent consolidative allogeneic haematopoietic cell transplantation (al-loHCT). [1][2][3] Unfortunately, many patients with ALL who receive CAR-T cells are ineligible for alloHCT due to failing prior transplant(s), co-morbidities or lack of donors. Tyrosine kinase inhibitor (TKI) therapy has improved the outcomes in Philadelphia chromosome-positive (Ph+) ALL and has been adopted as a maintenance therapy in alloHCT recipients.…”

The affinity of antigen-binding domain on the antitumor efficacy of CAR T cells: Moderate is better

“…Using CyTOF technology, Michelozzi et al. compared the FMC63 (high-affinity) and CAT (moderate-affinity) CD19 CARTs and found that, after engaging with CD19+ leukemia cells, the CAT CD19 CARTs contained significantly more polyfunctional T cells than the FMC63-derived CARTs ( 160 ). This suggests proper moderate-affinity ABD may allow CARTs to preserve their polyfunctionality, which is important for antitumor effect ( 161 ).…”

“…Such transient break during “off” time may rejuvenate and preserve CART function ( 37 ). Similarly, the moderate-affinity CAT mAb has similar K on as high-affinity FMC63, but has much higher K off ( 151 ), which may contribute to the formation of memory T cells and polyfunctionality of CAT CARTs ( 160 ) and durable antitumor effects ( 151 ). Thus, the dwell time and kinetics of ABD-target engagement may be more important than affinity (K D ) in deciding the outcome of CARTs.…”