Low CD19 Antigen Density Diminishes Efficacy of CD19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains
Authors: Majzner Robbie G. , Rietberg Skyler P. , Labanieh Louai , Sotillo Elena , Weber Evan W. , Lynn Rachel C. , Theruvath Johanna L. , Yuan Constance M. , Xu Peng , Nguyen Sang M. , Shah Nirali N. , Stetler-Stevenson Maryalice , Fry Terry J. , Lee Daniel W. , Mackall Crystal L. 
Published: October 2019

Abstract

Target antigen density has emerged as a major factor influencing the potency of CAR T cells. Our laboratory has demonstrated that the activity of numerous CARs is highly dependent on target antigen density (Walker et al., Mol Ther, 2017), and high complete response rates in a recent trial of CD22 CAR T cells for B-ALL were tempered by frequent relapses due to decreased CD22 antigen density on lymphoblasts (Fry et al., Nat Med, 2018). To assess if antigen density is also a determinant of CD19 CAR T cell therapeutic success, we analyzed CD19 antigen density from fifty pediatric B-ALL patients treated on a clinical trial of CD19-CD28ζ CAR T cells. We found that patients whose CD19 expression was below a threshold density (2000 molecules/lymphoblast) were significantly less likely to achieve a clinical response than those whose leukemia expressed higher levels of CD19.
In order to further understand this limitation and how it may be overcome, we developed a model of variable CD19 antigen density B-ALL. After establishing a CD19 knockout of the B-ALL cell line NALM6, we used a lentivirus to reintroduce CD19 and then FACS sorted and single cell cloned to achieve a library of NALM6 clones with varying CD19 surface densities. CD19-CD28ζ CAR T cell activity was highly dependent on CD19 antigen density. We observed decreases in cytotoxicity, proliferation, and cytokine production by CD19 CAR T cells when encountering CD19-low cells, with an approximate threshold of 2,000 molecules of CD19 per lymphoblast, below which, cytokine production in response to tumor cells was nearly ablated.
Given that a CD19-4-1BBζ CAR is FDA approved for children with B-ALL and adults with DLBCL, we wondered whether CARs incorporating this alternative costimulatory domain would have similar antigen density thresholds for activation. Surprisingly, CD19-4-1BBζ CAR T cells made even less cytokine, proliferated less, and had further diminished cytolytic capacity against CD19-low cells compared to CD19-CD28ζ CAR T cells. Analysis by western blot of protein lysates from CAR T cells stimulated with varying amounts of antigen demonstrated that CD19-CD28ζ CAR T cells had higher levels of downstream signals such as pERK than CD19-4-1BBζ CAR T cells at lower antigen densities. Accordingly, calcium flux after stimulation was also significantly higher in CD19-CD28ζ than CD19-4-1BBζ CAR T cells.
In a xenograft model of CD19-low B-ALL, CD19-4-1BBζ CAR T cells demonstrated no anti-tumor activity, while CD19-CD28ζ CAR T cells eradicated CD19-low leukemia cells. Therefore, the choice of costimulatory domain in CAR T cells plays a major role in modulating activity against low antigen density tumors. CD28 costimulation endows high reactivity towards low antigen density tumors. We confirmed the generalizability of this finding using Her2 CAR T cells; Her2-CD28ζ CAR T cells cleared tumors in an orthotopic xenograft model of Her2-low osteosarcoma, while Her2-4-1BBζ CAR T cells had no effect. This finding has implications for CAR design for lymphoma and solid tumors, where antigen expression is more heterogeneous than B-ALL.
To enhance the activity of CD19-4-1BBζ CAR T cells against CD19-low leukemia, we designed a CAR with two copies of intracellular zeta in the signaling domain (CD19-4-1BBζζ). T cells expressing this double-zeta CAR demonstrated enhanced cytotoxicity, proliferation, cytokine production, and pERK signaling in response to CD19-low cells compared to single-zeta CARs. Additionally, in a xenograft model, CD19-4-1BBζζ CAR T cells demonstrated enhanced activity against CD19-low leukemia compared to CD19-4-1BBζ CAR T cells, significantly extending survival. The addition of a third zeta domain (CD19-4-1BBζζζ) further enhanced the activity of CAR T cells. However, inclusion of multiple copies of the costimulatory domains did not improve function.
In conclusion, CD19 antigen density is an important determinant of CAR T cell function and therapeutic response. CD19-CD28ζ CARs are more efficient at targeting CD19-low tumor cells than CD19-4-1BBζ CARs. The addition of multiple zeta domains to the CAR enhances its ability to target low antigen density tumors. This serves as proof of concept that rational redesign of CAR signaling endodomains can result in enhanced function against low antigen density tumors, an important step for extending the reach of these powerful therapeutics and overcoming a significant mechanism of tumor escape.

Disclosures
Lee: Juno: Consultancy.