Modulation of CD22 Antigen Density Improves Efficacy of CD22 Chimeric Antigen Receptor (CAR) T Cells Against CD22^lo B-Lineage Leukemia and Lymphoma

While overall survival for acute lymphoblastic leukemia (ALL) in children treated with risk-adapted, multi-agent chemotherapeutic regimens is greater than 85%, relapsed ALL remains a major challenge. Chimeric antigen receptor (CAR) T cell therapy targeting CD19 provides these patients with a remission induction rate of 70-90% across multiple clinical trials, but loss of the targeted epitope results in relapse following CD19 CAR T cells. In the setting of these relapses, our group conducted a CD22 CAR T cell trial, which induces remission in approximately 70% percent of patients with relapsed/refractory CD22-positive pre-B cell ALL, including those relapsing after CD19 CAR therapy (NCT02315612). However, relapse associated with reduced CD22 site density has shortened remission duration in some patients. To test the impact of low CD22 site density on CD22 CAR T cell functionality, we modified Nalm6 ALL cell lines using CRISPR/Cas9 to eliminate the CD22 gene, followed by lentiviral transduction of full-length CD22 and single-cell cloning based on CD22 expression levels. CD22 site density was assessed with the Quantibrite-PE Assay Kit and four cell lines were identified for a spectrum of CD22 expression: CD22 negative (CD22^neg); CD22 low (CD22^lo) - site density comparable to that seen in ALL patients relapsing following CD22 CAR therapy; CD22 normal (Nalm6) - site density comparable to ALL CD22 expression; and CD22 high (CD22^hi) - site density comparable to CD19 levels. As CD22 site density decreases, CD22 CAR T cells produce incrementally less interferon-gamma and do not produce IL-2 against CD22^lo ALL. CD22 CAR delayed, but was unable to prevent, progression of CD22^lo ALL in murine xenografts. We next evaluated two potential approaches to overcoming low site density antigen escape: improving CAR activity or increasing tumor sensitivity. To improve CAR activity, we generated a high-binding affinity CD22 CAR (CD22 CAR^H1) using an affinity-enhanced version of the scFv from the CD22 CAR construct in our ongoing trial. Despite higher affinity, the CD22 CAR^H1 T cell did not improve in vitro killing or in vivo clearance of CD22^lo ALL. To increase tumor sensitivity, we sought to increase CD22 site density using the PKC inhibitor Bryostatin1, a drug previously shown to increase CD22 expression in CLL. Bryostatin1 increases CD22 site density on a panel of pre-B cell ALL and diffuse large B cell lymphoma (DLBCL) cell lines. Interestingly, the CD22 increase is durable for at least 1 week after exposure to Bryostatin1. Bryostatin1-mediated CD22 increased site density improves cytokine production by CD22 CAR T cells. To evaluate the mechanism of Bryostatin1-mediated upregulation of CD22 expression, RNAseq was performed on Bryostatin1-exposed ALL. Preliminary analysis demonstrates that Bryostatin1 does not directly affect CD22 mRNA expression levels, but modulates early transcription factors within the B-cell receptor pathway. Based on these results, we are further evaluating pathways affected by Bryostatin1 through protein expression analysis and assessing in vivo function of Bryostatin1 in the setting of a patient-derived CD22 CAR-resistant xenograft model. In conclusion, these results demonstrate a clear threshold antigen site density, below which the CD22 CAR is unable to clear CD22-expressing ALL. We further demonstrate that increased CD22 CAR affinity does not seem to improve CD22 CAR activity against CD22^lo ALL. Bryostatin1-mediated CD22 modulation can improve CD22 CAR response against leukemia and lymphoma providing a potential rational combination to rescue CD22 CAR activity and remission durability.