Juno Therapeutics is developing Chimeric Antigen Receptor (CAR) T-cells directed against B-cell antigen CD19 for the treatment of patients with B-cell lymphomas. The company has elected to halt the development of JCAR015 for Acute Lymphoblastic Leukemia (ALL) and proceed with JCAR017 for relapsed/refractory diffuse large B-cell lymphoma (DLBCL), the most common form of Non-Hodgkin Lymphoma (NHL), due to the development of cerebral edema and subsequent death of several patients with ALL enrolled in its phase 2 ROCKET trial, which has been suspended.
Chimeric Antigen Receptor T-cells
CAR T-cells have evolved from first generation constructs that contained an extracellular binding domain and intracellular signaling domain, to third generation structures that also include co-stimulatory molecules.
JCAR015 and JCAR017 both target the CD19 antigen, a 95 kilodalton glycoprotein belonging to the immunoglobulin class. No significant homology exists between CD19 and other known proteins, making it an ideal biomarker for B-cells.
CD19 acts as a critical co-receptor for BCR signal transduction. As BCR (B-cell receptor) signaling requires protein tyrosine kinase (PTK) activation, CD19 recruits and amplifies the activation of Src-family protein tyrosine kinases such as Lyn and Fyn. Upon BCR activation, CD19 also enhances BCR-induced signaling crucial for B cell expansion, through recruitment and activation of PI3K and downstream Akt kinases.
JCAR015 versus JCAR017
These Chimeric Antigen Receptors are comprised of four domains: (1) extracellular targeting – single chain variable fragment; (2) transmembrane; (3) cytoplasmic costimulatory; and (4) cytoplasmic signaling domain.
Upon recognition and binding of the scFv of the CAR T cell to the cancer cell, there is a conformational change that leads to an activation signal to the cell through CD3-zeta, an intracellular signaling protein. Our current CAR constructs also include either a CD28 or 4-1BB costimulatory signaling domain to mimic a “second signal” that amplifies the activation of the CAR T cells, leading to a more robust signal to the T cell to multiply and kill the cancer cell.
There are important differences between JCAR015 and JCAR017, which likely account for the improved safety profile of the latter:
- The cells used to create JCAR015 are composed of CD3+ enriched peripheral blood mononuclear cells, whereas JCAR017 is built using a fixed ratio of CD4+ and CD8+ T-lymphocytes.
- Each agent is engineered using different viral vectors, namely gamma retroviral for JCAR015 and lentiviral for JCAR017.
- The binding domain for JCAR015 is SJ25C1 and the costimulatory domain is CD28, whereas JCAR017 has a binding domain of FMC63 and a costimulatory domain of 4-1BB.
- JCAR017 contains an ablative technology to provide better control of proliferation and survival of the engineered T cells, which is not included in JCAR015. This ablative technology, a truncated form of the human epidermal growth factor receptor (EGFRt), allows for rapid killing of the CAR T-cells using cetuximab (Erbitux), if needed. The addition of EGFRt may also have immunostimulating properties.
By including only CD4+ (T-helper) and CD-8+ (Cytotoxic T-cells), the immune attack is more focused – CD3+ positive cells also include macrophages. Co-stimulatory molecule CD28 stimulates the B7 signaling pathway, which results in stabilization of cytokine mRNA, whereas, 4-1BB triggers the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) pathway, which results activation of NFkB . CAR T-cells with CD28 expand rapidly, whereas, those with 4-1BB expand more slowly – this is thought to contribute to improved tolerability, including less cerebral edema.
CAR T-cells incorporating 4-1BB are more persistent and less likely to undergo exhaustion than those incorporating CD28. This probably contributes to improved effectiveness seen with CAR017. Inclusion of EGFR in the construct adds a great measure of safety – the ability to neutralize the CAR T-cells is critical should adverse events emerge.
Clinical results with JCAR017
JCAR017 has shown impressive results in the clinic to data. In a phase 1 study in patients with NHL, a 60% complete response (CR) rate and an 80% overall response rate were observed. None of the patients experienced severe cytokine release syndrome (CRS) and fourteen percent experienced neurotoxicity, which resolved with treatment.
In a phase 1 study of young patients with relapsed/refractory CD-19 positive ALL, the minimal residual disease (MRD)-negative CR rate was 93%. In those pre-treated with fludarabine and cyclophosphamide, the MRD-negative CR rate was 100%. Twenty-three percent experienced severe CRS, and grade 3 neurotoxicity was seen in 23% of patients.
In adults with ALL, the CR rate was 77% and the MRD-negative rate was 90% (in those who could be evaluated for MRD). Twenty-seven percent of patients experienced severe CRS, and twenty-nine percent had grade 3 or 4 neurotoxicity.