Maciocia, Nicola Christina; (2022) Anti-CD21 Chimeric Antigen Receptor Therapy for the Treatment of T-cell Acute Lymphoblastic Leukaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Relapsed/refractory (r/r) T cell acute lymphoblastic leukaemia (T-ALL) has a dismal prognosis, with an unmet need for effective novel therapies. Several unique challenges mean that the success seen in chimeric antigen receptor (CAR)-T cell therapy for B cell malignancies has yet to be successfully translated to T-ALL. Most strategies have targeted pan-T cell antigens (CD7, CD5) but these may be limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen surface expression during manufacture and salvage haematopoetic stem cell transplantation (HSCT). An ideal CAR-T target would be exclusively or largely confined to the malignant T cell component but published examples of these (CD1a and TRBC1) are expressed in only a minority of cases of T-ALL. This thesis proposes a novel immunotherapeutic strategy for the treatment of T-ALL. We have developed the little-known T cell target, CD21, as a target for CAR-T. CD21 is expressed in T-ALL in a NOTCH-dependent manner with normal expression restricted to B cells and follicular dendritic cells (FDCs), and a minor fraction of mature T cells. We initially sought to validate CD21 as a potential target. 70% of human T-ALL cell lines (11/16) expressed surface CD21 by flow cytometry (FACS), with a mean antigen density in positive lines of 2569 copies/cell. In primary T-ALL, 57% of presentation samples (n=58) expressed CD21 (mean antigen density 1262 copies/cell). 45% of relapse (n=11) and 20% of primary refractory cases (n=30) expressed CD21, with a similar antigen density to presentation samples. CD21 positivity varied by maturational stage, with highest expression in cortical T-ALL (80% of cases) followed by pre-T (72%), mature (67%), early thymocyte precursor (ETP) (25%) and pro-T (17%). Healthy donor blood (n=14) showed CD21 expression limited to B cells and a low proportion (11%) of T cells (316 copies/cell). T cell CD21 expression was not up-regulated upon activation (n=6), was highest in naïve and gamma-delta subsets and was not associated with markers of differentiation/ exhaustion. To target CD21, we performed DNA gene-gun vaccination of rats with a plasmid encoding full-length CD21, followed by phage display, and isolated multiple anti-CD21 scFvs which were all found to bind to membrane-distal CD21 epitopes. These were cloned into 4-1BBζ second-generation CARs and expressed in primary T cells but failed to kill or secrete cytokines in response to CD21+ SupT1 cells. CD21 has a bulky extracellular domain containing 15-16 sushi repeats. We hypothesized that anti-CD21 CAR-T cells binding to membrane-proximal epitopes would form more efficient immunological synapses and signal more effectively. We re-vaccinated rats with the 5 most membrane-proximal sushi repeats of CD21 and generated a collection of binders through phage display and hybridoma which bound CD21 at this membrane-proximal region. Many of these CD21 proximal-binding CARs were functional, with cytotoxicity and interferon-γ secretion against CD21+ target cells. However, non-specific cytokine secretion was seen against CD21-negative cells, and no IL-2 secretion was seen. Multiple engineering approaches were tested to increase potency and specificity of these CARs. Re-cloning binders into a fragment antigen binding (Fab)-CAR architecture yielded constructs capable of specific cytotoxicity, IFN-γ and IL2 release against a CD21+ cell line. Our lead anti-CD21 candidate CAR proliferated in vitro, without fratricide or premature exhaustion/ differentiation, and was active against low-density CD21-positive cell lines (n=3) and primary cells from 2 T-ALL patients. We tested anti-CD21 CAR in two cell line xenograft murine models of T-ALL. NSG mice were injected with SUPT1-luciferase and MOLT4-luciferase cells and treated with CAR19 or CAR21 cells. In both models, lower disease burden was seen in CD21 CAR-T v CD19 recipients by bioluminescence imaging. We injected primary T-ALL blasts in two further murine cohorts, treating again with CAR19 or CAR21. Serial bleeds post CAR-T showed significant tumour control in CAR21 treated mice compared with CAR19 treated mice translating to an overall survival advantage in both models. Finally, through exploiting knowledge of its NOTCH1 mediated expression, we have been able to modulate CD21 antigen density pharmacologically using PI3K-AKT-mTOR inhibitors and have demonstrated that an increase in CD21 surface expression results in improved in vitro CAR21 functionality. In summary, we propose CD21 as a novel target for CAR-T cell therapy in T-ALL. Its expression is largely restricted to the malignant T cell compartment, overcoming issues with fratricide and on-target off-tumour effects seen in many T-ALL CAR-T strategies to date. Despite the complexity of the target, we have successfully generated an CD21 targeting CAR that is functional both in vitro and in vivo.

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