Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

J Immunother Cancer. 2020 Aug;8(2):e000896. doi: 10.1136/jitc-2020-000896.


Background: There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19- either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19- B-ALL relapses and CD19- preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.

Methods: Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.

Results: Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.

Conclusions: We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

Keywords: T-lymphocytes; cell engineering; hematologic neoplasms; immunotherapy; receptors, chimeric antigen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Epitopes / genetics*
  • Humans
  • Male
  • Mice
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma / genetics*
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma / pathology
  • Receptors, Chimeric Antigen / immunology*
  • Sialic Acid Binding Ig-like Lectin 2 / metabolism*


  • CD22 protein, human
  • Epitopes
  • Receptors, Chimeric Antigen
  • Sialic Acid Binding Ig-like Lectin 2