Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

Abstract

Key Points• We have created a new highly active chimeric antigen receptor (CAR) specific for CD22.• The design of new CARs may benefit more from target antigen epitope selection than from optimizing affinity.Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ؎ an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. (Blood. 2013;121(7):1165-1174) IntroductionDespite great progress in the treatment of children and adults with acute lymphoblastic leukemia (ALL), substantial numbers of patients continue to die of this disease and the short and long-term toxicities of standard therapy are substantial. 1-3 Monoclonal antibody-based therapies offer promise for overcoming chemoresistance and potentially diminishing the toxicities associated with therapy. 4 Among the most promising of these therapies involve the engineering of mature T lymphocytes to recognize MHC nonrestricted tumor antigens by transducing chimeric antigen receptors (CARs), reviewed by Lee at al. 5 CARs incorporate an extracellular binding domain (often derived from the antigen binding region of an antibody) with transmembrane and signaling motifs to render T cells capable of targeting any surface antigen that is amenable to antibody-like recognition. Early clinical results have demonstrated impressive antitumor effects in patients with leukemia, 6-10 although the ideal CAR design with respect to structural and signaling features remains unclear and has been the topic of intense inquiry.B-cell antigens are compelling targets for CAR-based therapies because normal tissue expression of these antigens is restricted to the B-cell lineage and clinical tolerance for B-cell ablation is high using modern supportive care. Indeed, CARs targeting CD19 have demonstrated activity against B-cell malignancies with acceptable toxicity 6-8 as have anti-CD20 antibodies in CD20 ϩ malignancies, including CD20-expressing ALL. 11 CD22 is another member of the B-cell antigen family with a tis...

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…The low EC 50 value likely reflects the extremely high potency of blinatumomab. 19 Of note, TBEPC R was calculated based on the experimentally determined expression values of T cell CD3 and Nalm-6 cell CD19 and the reported blinatumomab binding affinities to CD3 and CD19 on cell surfaces following 45-minute incubations; 22,23 any potential divergence between the assay measurements and true values of receptor density and binding affinity would affect the derivation of TBEPC R values and the estimation of EC 50 . The baseline killing in the absence of the drug (Base) was fixed at 0, based on the observed data.…”

“…The effect of T cell proliferation was not considered in the current simulation since it was reported that, on average, blinatumomab caused approximately a two-fold expansion of T cells in patients, 41 which would only lead to minor changes in τ. CD19 expression level on B cells was assigned at 20,000 receptors per cell based on literature report, 42 and this value was 2- to 5-fold lower than that of B cell lines used in vitro cytotoxicity assays (20,000 vs. 40,000 – 100,000 receptors per cell). 22,33,43–45 The blood and bone marrow blinatumomab concentration-response curves predicted by the TBE model are shown in Figure 5. Blinatumomab plasma drug concentration information following the approved dosing regimen, i.e., continuous intravenous infusion at a 9 µg/day priming dose for 7 days and then 28 µg/day full dose treatments, was obtained from multiple clinical PK studies in patients with either ALL or non-Hodgkin’s lymphoma.…”

“…22,33,43,44 CD19 expression levels on B cells was assigned at 20,000 receptors per cell based on data reported in the literature. 42 The expression level of CD33 on parental OCI-AML3 cells was 500, and the expression levels of CD33 on OCI-AML3 cells transduced with low, medium, and high levels of CD33 were 1,500, 5,000, and 10,000 receptors per cell, respectively, obtained by digitization.…”

Recent advances in T‐cell immunotherapy for haematological malignancies

“…(Sotillo et al 2015) Another potential solution for antigen escape is targeting multiple surface antigens on a single tumour in one product, as described in the Tumour Associated Antigen section above. Until recently, CD22-CAR targeted therapy was limited to the pre-clinical realm(Haso et al 2013). However, in addition to targeting CD22 in patients with CD19 antigen loss following CD19-directed therapy (NCT02315612), several groups are currently exploring the use of bispecific CAR T-cells in preclinical studies.…”

Chimeric antigen receptor modified T cell therapy for B cell malignancies

“…Multiple cell surface antigens expressed by B cell malignancies, including CD19, CD20, CD22, CD23, CD38, ROR1, and kappa light chain, have been targeted with T cells engineered to express scFv-containing CAR constructs [13,15,[20][21][22][23][24][25], and some of these antigens are now being targeted in phase I clinical trials of CAR-modified T cell therapy (Table 1). Although targeting using a scFv incorporated into a CAR has remained the most prevalent strategy to target B cell malignancies, other approaches have been used to target CAR-modified T cells to solid tumors, for example by incorporation of IL-13 into a CAR to redirect engineered T cells to IL-13Ra2-expressing tumors [26].…”